Methods of selectively forming substituted pyrazines

ABSTRACT

Methods of selectively forming substituted pyrazines are provided. Methods of the present invention can include receiving a reaction solution including at least one carbon source and at least one nitrogen source, and heating the reaction solution to a reaction temperature and holding the reaction solution at the reaction temperature for a time sufficient to produce a reaction product comprising at least one substituted pyrazine. The carbon source can be selected from the group consisting of hydroxy ketone(s), sugar(s) treated with at least one buffer, and combinations thereof. Tobacco products incorporating substituted pyrazines are also provided.

FIELD OF THE INVENTION

The present invention relates to methods of forming substitutedpyrazines. Of particular interest are methods of selectively formingtargeted substituted pyrazines.

BACKGROUND OF THE INVENTION

Pyrazines are produced by reactions (e.g., Maillard) of a carbon sourcewith nitrogenous compounds, such as amino acids and bases (e.g.,diammonium phosphate (DAP), NaOH). In many conventional reactionpathways intended to produce pyrazines, a sugar (e.g., fructose,glucose, fructose/glucose mixtures, rhamnose) is used as the carbonsource. To date, the vast majority of model reactions and fortifiednatural products which result in pyrazine-rich formulations upon heatinghave used sugars such as fructose, glucose, fructose/glucose mixtures,and rhamnose as the carbon source components of the formulations. Thesesugars have been shown to serve as carbon sources for the formation ofthe pyrazine aromatic ring structure. See, e.g., U.S. Pat. App. Pub.Nos. 2004/0173228 to Coleman, 2010/0037903 to Coleman et al.,2012/0152265 to Dube et al., 2012/0260929 to Coleman et al.,2013/0125907 to Dube et al., 2013/0337418 to Anuradha, 2015/0040922 toDube et al., and 2015/0122271 to Chen et al.; U.S. Pat. No. 5,258,194 toAnderson et al., U.S. Pat. No. 6,298,858 to Coleman; U.S. Pat. No.6,325,860 to Coleman, U.S. Pat. No. 6,440,223 to Dube et al., U.S. Pat.No. 6,499,489 to Coleman; U.S. Pat. No. 6,591,841 to White et al., U.S.Pat. No. 6,695,924 to Dube et al., U.S. Pat. No. 8,434,496 to Chen etal., U.S. Pat. No. 8,944,072 to Brinkley et al., U.S. Pat. No. 8,955,523to Coleman et al., U.S. Pat. No. 8,991,403 to Chen et al., U.S. Pat. No.9,010,339 to Dube et al., U.S. Pat. No. 9,254,001 to Byrd et al., U.S.Pat. No. 9,265,284 to Junker et al., and U.S. Pat. No. 9,402,415 toColeman et al.; and Coleman III, On the synthesis and characteristics ofaqueous formulations rich in pyrazines, in Flavor Fragrance and OdorAnalysis, Second Edition, Ray Marsili, ed., Chapter 7, pp 135-182, CRCPress, Boca Raton, 2012; each of which is herein incorporated byreference in its entirety.

Most often, these reactions with sugar(s) and a nitrogen source haveemployed ammonium hydroxide and/or free amino acids as nitrogen sourcesthat furnish the nitrogen bond within the pyrazine structure. See, e.g.,Effect of time, temperature, and reactant ratio on pyrazine formation inmodel system, T. Shibamoto, R. A. Bernhard, J. Agric. Food Chem., 24,(1976) p. 847. The reaction produces a complex mixture of manysubstituted pyrazines. When a sugar serves as the sole carbon source inthe reaction to produce pyrazines, the molecules pyrazine andmethylpyrazine are the dominant pyrazines produced, often much greaterthan 60% of the total pyrazine yield. Even when free amino acids areemployed as co-reagents in an attempt to reduce the amount of pyrazineand methylpyrazine molecules produced, this trend is evident.

From a sensory perspective, the molecules pyrazine and methylpyrazinepossess neither desirable sensory notes nor acceptable volatilitycharacteristics for use in tobacco products. Thus, their presence in amixture of pyrazines is a less than desirable characteristic for certainapplications.

As such, it would be desirable to provide methods for selectivelyproducing certain desirable substituted pyrazines in greater amounts.

SUMMARY OF THE INVENTION

The present invention provides methods of selectively forming certainsubstituted pyrazines. A method of selectively producing pyrazines cancomprise receiving a reaction solution comprising at least onetobacco-derived carbon source (e.g., a hydroxy ketone and/or at leastone sugar treated with a buffer) and at least one tobacco-derivednitrogen source (e.g., a protein and/or an amino acid), and heating thereaction solution to a reaction temperature and holding the reactionsolution at the reaction temperature for a time sufficient to produce areaction product comprising at least one substituted pyrazine. Thenitrogen source can be selected from the group consisting of aminoacids, ammonium ions, and combinations thereof.

In various embodiments, the at least one substituted pyrazine can beselected from the group consisting of 2,6-dimethylpyrazine;2,5-dimethylpyrazine; 2-ethyl-5-methylpyrazine;2-ethyl-6-methylpyrazine; 2,3,5-trimethylpyrazine;2-ethyl-3,5-dimethylpyrazine; 2-ethyl-2,5-dimethylpyrazine;2,3,5,6-trimethylpyrazine; 2,3,5-trimethyl-6-ethylpyrazine;2,6-dimethyl-3-propylpyrazine; 2,5-diethyl-3,6-dimethylpyrazine;2,6-dimethyl-3-(2-methylbutyl)pyrazine;2,5-dimethyl-3-(2-methylbutyl)pyrazine;2,5-dimethyl-3-(3-methylbutyl)pyrazine; 2,5-dimethyl-3-propylpyrazine;2,5-dimethyl-3-cis-propenylpyrazine; 2-isopropenyl-3,6-dimethylpyrazine;2-(2-methylpropyl)-3,5-dimethylpyrazine;2,6-dimethyl-3-isobutylpyrazine;2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine;trimethylpyrazine; furaneol, 2,5-dimethyl-3-ethylpyrazine;tetramethylpyrazine; 2,3-diethyl-5-methylpyrazine;2,5-dimethyl-3-propenylpyrazine; 2,3,5-trimethyl-6-isopropylpyrazine;2-acetyl-4,5-dimethylpyrazine; 3,5-dimethyl-2-methylpropylpyrazine;2,6-diethylpyrazine; 2,5-diethylpyrazine; 2-ethyl-3,5,6-trimethylpyrazine; 3.5-dimethyl-2-(n-propyl)pyrazine;3,6-dimethyl-2-(n-propyl)pyrazine; 2,5-diethyl-3-methylpyrazine;2,3-diethyl-5,6-dimethylpryazine;trans-3-methyl-2-(n-propyl)-6-(butenyl)pyrazine;2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrazine; and2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

In some embodiments, the at least one substituted pyrazine isdisubstituted, trisubstituted, or tetrasubstituted. In certainembodiments, the at least one substituted pyrazine comprises at leastone substituent group having 2 or more carbon atoms. In variousembodiments, the at least one substituted pyrazine comprises at leastone substituent group having 3 or more carbon atoms.

In various embodiments, the method of selectively forming certainsubstituted pyrazines can further comprise isolating the at least onesubstituted pyrazine from the reaction product. The step of isolatingthe at least one tobacco-derived pyrazine from the reaction product cancomprise at least one of liquid-liquid extraction of the reactionproduct, liquid-solid extraction of the reaction product, and simpledistillation of the reaction product, for example.

The methods of the present invention can further comprise incorporatingat least one substituted pyrazine into a tobacco product. In certainembodiments, the tobacco product can be a smoking article. In someembodiments, the tobacco product can be a smokeless tobacco product.

In various embodiments of the present invention, a method of formingpyrazines is provided, the method comprising receiving a reactantsolution comprising at least one alpha-hydroxy ketone and at least onenitrogen source, and heating the reactant solution to a reactanttemperature and holding the reactant solution at the reactanttemperature for a time sufficient to produce a reactant productcomprising at least one substituted pyrazine. Various embodiments of themethod can further comprise isolating the at least one substitutedpyrazine from the reactant product. In certain embodiments, the at leastone hydroxy ketone can comprise acetol and the at least one substitutedpyrazine can be selected from the group consisting of:2,3-dimethylpyrazine; 2,6-dimethylpyrazine; 2-ethyl-6-methylpyrazine;2-ethyl-5-methylpyrazine; trimethylpyrazine; furaneol;2,5-dimethyl-3-ethylpyrazine; 2-ethyl-3,5-dimethylypyrazine;tetramethylpyrazine; 2,5-dimethyl-3-propenylpyrazine;2,3,5-trimethyl-6-isopropylpyrazine; 2-acetyl-4,5-dimethylpyrazine;3,5-dimethyl-2-methylpropylpyrazine, and combinations thereof.

In some embodiments, the at least one hydroxy ketone can compriseacetoin and the at least one substituted pyrazine can betetramethylpyrazine. In various embodiments, the at least one hydroxyketone can comprise 1-hydroxy-2-butanone and the at least onesubstituted pyrazine can be selected from the group consisting of:2,6-diethylpyrazine; 2,5-diethylpyrazine;2-ethyl-3,5,6-trimethylpyrazine; 3,5dimethyl-2-(n-propyl)pyrazine;3,6-dimethyl-2-(n-propyl)pyrazine; 2,5-diethyl-3-methylpyrazine;2,3-diethyl-5-methylpyrazine; 2,3-diethyl-5,6-dimethylpryazine;trans-3-methyl-2-(n-propyl)-6-(butenyl)pyranzine; ;2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

In various embodiments of the present invention, the method can furtherinclude adding free amino acids to the reaction solution comprising atleast one alpha-hydroxy ketone and at least one nitrogen source. In someembodiments, the method can further include adding at least one aldehydeto the reaction solution comprising at least one alpha-hydroxy ketoneand at least one nitrogen source.

In various embodiments of the present invention, a method of formingpyrazines is provided, the method comprising receiving a carbon sourcesolution comprising at least one sugar and at least one buffer such thatan optimized amount of at least one hydroxy ketone is provided from theat least one sugar, mixing the carbon source solution with at leastnitrogen source to form a reaction solution, and heating the reactionsolution to a reaction temperature and holding the reaction solution atthe reaction temperature for a time sufficient to produce a reactionproduct comprising at least one substituted pyrazine. Variousembodiments of the methods herein can further comprise isolating the atleast one substituted pyrazine from the reaction product. The at leastone sugar can be selected from the group consisting of glucose,fructose, rhamnose, and combinations thereof. In certain embodiments,the buffer can be selected from the group consisting of: sodiumhydroxide, a phosphate buffer, and combinations thereof. In certainembodiments, the buffer can buffer in a pH range of about 6.5 to about7.5. Methods of the present invention can further comprise addingammonium ions to the reaction solution comprising a carbon sourcecomprising at least one sugar and at least one buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to provide an understanding of embodiments of the invention,reference is made to the appended drawings, which are not necessarilydrawn to scale, and in which reference numerals refer to components ofexemplary embodiments of the invention. The drawings are exemplary only,and should not be construed as limiting the invention.

FIG. 1 is a flow chart describing methods of selectively formingsubstituted pyrazines;

FIG. 2 is a flow chart describing methods of selectively formingsubstituted pyrazines;

FIG. 3 shows the GC/MS analysis of glucose reacted with a phosphatebuffer at 140° C. for 60 min and extracted with dichloromethane (DCM);

FIG. 4 shows the GC/MS analysis of pyrazines extracted using DCM from areaction mixture of (1 gram of glucose reacted with 25 mL of 40%phosphate buffer at 140° C. for 60 min) with 1 mL of NH₄OH at 140° C.for 17 hours;

FIG. 5 shows the GC/MS analysis of pyrazines extracted using DCM from 25mL of a reaction mixture of (0.1 N NaOH reacted with 0.5 gram of glucoseat 140° C. for 60 min) reacted with 1 mL of NH₄OH at 140° C.;

FIG. 6 is an exploded perspective view of a smoking article having theform of a cigarette, showing the smokable material, the wrappingmaterial components, and the filter element of the cigarette;

FIG. 7 is a top view of a smokeless tobacco product embodiment, takenacross the width of the product, showing an outer pouch filled with atobacco material; and

FIG. 8 is a sectional view through an electronic smoking articlecomprising a cartridge and a control body and including a reservoirhousing according to an example embodiment of the present disclosure.

DETAILED DESCRIPTION

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. As used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Reference to “dry weight percent” or“dry weight basis” refers to weight on the basis of dry ingredients(i.e., all ingredients except water).

The present invention provides methods of forming selected pyrazines.Pyrazines display many different flavor profiles, including, but notlimited to, roasted, toasted and nutty notes. For example, pyrazineswith cyclopentyl derivatives are known for their positive sensoryattributes at very low levels, ppb. Pyrazines are formed by heatingmixtures of carbon sources and nitrogen sources. Methods of the presentinvention not only minimize the formation of the molecules pyrazine andmethylpyrazine, but also tailor the reaction so that other desiredsubstituted pyrazines can be produced in a controlled fashion.

Selective Formation of Pyrazines Using a Carbon Source Other thanSugar(s)

Conventionally, sugar(s) have been used as the carbon source inreactions to form pyrazines. Several reaction pathways are known in theart to produce pyrazine-rich formulations using sugar(s), including: 1)hydrolysis of protein into free amino acids followed by reaction ofthose free amino acids with sugar(s) such as glucose and/or highfructose syrup; and 2) biotechnical synthesis of free amino acidsemploying glucose and nitrogen (e.g., ammonium ions) followed byreaction of these free amino acids with sugar(s) such as glucose and/orhigh fructose syrup. When sugars are employed as an intact molecule andreacted with a nitrogen source (e.g., ammonium hydroxide, amino acids),an array of pyrazines are produced, including the molecules pyrazine andmethylpyrazine as the dominate pyrazines, with much lesser amounts ofdimethylpyrazines, and significantly less amounts of higher molecularweight pyrazines.

As used herein, the term “the molecule pyrazine” refers to aheterocyclic organic compound with the chemical formula C₄H₄N₂. This isdifferent from the general term “pyrazine(s)” used herein, which refersto the group of compounds produced from the reaction of a carbon sourcewith a nitrogen source.

As used herein, the term “nitrogen source” refers to a nitrogencontaining compound that is reactive with a carbon source to form atleast one pyrazine. In various embodiments, a nitrogen source cancomprise ammonium ions (NH₄ ⁺), amino acids, proteins, or a combinationthereof. In some embodiments, ammonium ions (NH₄ ⁺) can be provided bycompounds such as ammonium hydroxide, diammonium phosphate (DAP), etc.Amino acids can be derived from hydrolysis of protein, for example. Insome embodiments, Amino acids can be derived from hydrolysis oftobacco-derived protein, as discussed in U.S. patent application Ser.No. 15/009,199 to Dube et al. filed Jan. 28, 2016, which is hereinincorporated by reference in its entirety. Other compounds containingnitrogen that are known in the art and are reactive with a carbon sourceto form at least one pyrazine can also be used as nitrogen sources inembodiments of the invention disclosed herein.

In various embodiments of the present invention, the carbon source cancomprise a hydroxy ketone. A hydroxy ketone is a functional group of aketone flanked by a hydroxyl group. In two main classes of hydroxyketones, the hydroxyl group can be placed in the alpha position (i.e.,an alpha-hydroxy ketone having the formula RCR′(OH)(CO)R), or in thebeta position (i.e., a beta-hydroxy ketone having the formulaRCR′(OH)CR₂(CO)R). The structures of alpha and beta hydroxy ketones areillustrated below.

In various embodiments of the present invention, the carbon source cancomprise at least one alpha-hydroxy ketone. In various embodiments ofthe present invention, R and R′ functional groups of the at least onehydroxy ketone can be H or a substituent independently selected from thegroup consisting of halo (e.g., Cl, F, or Br), OH, optionallysubstituted C1-10 alkyl, optionally substituted C1-10 alkoxy, optionallysubstituted C2-4 alkenyl, optionally substituted C2-4 alkynyl, NR₆R₇,NR₆COR₇, NR₆CO₂R₇, CR₆R₇OR₈, CONR₆R₇, CO₂R₆, CN, CF₃, NO₂, N₃, C1-3alkylthio, R₉SO, R₉SO₂, CF₃S, and CF₃SO₂. In certain embodiments of thepresent invention, R and/or R′ functional groups are a C1-6 alkyl.

The term “alkyl” as used herein means saturated straight, branched, orcyclic hydrocarbon groups (i.e., cycloalkyl groups), as well asunsaturated versions of the saturated examples (e.g., propenyl). Inparticular embodiments, alkyl refers to groups comprising 1 to 10 carbonatoms (“C1-10 alkyl”). In further embodiments, alkyl refers to groupscomprising 1 to 8 carbon atoms (“C1-8 alkyl”), 1 to 6 carbon atoms(“C1-6 alkyl”), or 1 to 4 carbon atoms (“C1-4 alkyl”). In otherembodiments, alkyl refers to groups comprising 3-10 carbon atoms (“C3-10alkyl”), 3-8 carbon atoms (“C3-8 alkyl”), or 3-6 carbon atoms (“C3-6alkyl”). In specific embodiments, alkyl refers to methyl,trifluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl,t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl.

“Optionally substituted” in reference to a substituent group refers tosubstituent groups optionally substituted with one or more moietiesselected from the group consisting of, for example, halo (e.g., Cl, F,Br, and I); alkyl (e.g., C1-10 alkyl), halogenated alkyl (e.g., CF₃,2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃, or CF₂CF₃); C2-4 alkenyl, C2-4 alkynyl;hydroxyl; amino; amido; carboxylate; carboxamido; carbamate; carbonate;urea; acetate; alkylamino; arylamino; C1-10 alkoxy; aryl; aralkyl,aryloxy; nitro; azido; cyano; thio; alkylthio; sulfonate; sulfide;sulfinyl; sulfo; sulfate; sulfoxide; sulfamide; sulfonamide; phosphonicacid; phosphate; and/or phosphonate.

The term “alkenyl” as used herein means alkyl moieties wherein at leastone saturated C—C bond is replaced by a double bond. In particularembodiments, alkenyl refers to groups comprising 2 to 10 carbon atoms(“C2-10 alkenyl”). In further embodiments, alkenyl refers to groupscomprising 2 to 8 carbon atoms (“C2-8 alkenyl”), 2 to 6 carbon atoms(“C2-6 alkenyl”), or 2 to 4 carbon atoms (“C2-4 alkenyl”). In specificembodiments, alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl,4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl.

The term “alkynyl” as used herein means alkyl moieties wherein at leastone saturated C—C bond is replaced by a triple bond. In particularembodiments, alkynyl refers to groups comprising 2 to 10 carbon atoms(“C2-10 alkynyl”). In further embodiments, alkynyl refers to groupscomprising 2 to 8 carbon atoms (“C2-8 alkynyl”), 2 to 6 carbon atoms(“C2-6 alkynyl”), or 2 to 4 carbon atoms (“C2-4 alkynyl”). In specificembodiments, alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl,1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl.

The term “alkoxy” as used herein means straight or branched chain alkylgroups linked by an oxygen atom (i.e., —O-alkyl), wherein alkyl is asdescribed above. In particular embodiments, alkoxy refers tooxygen-linked groups comprising 1 to 10 carbon atoms (“C1-10 alkoxy”).In further embodiments, alkoxy refers to oxygen-linked groups comprising1 to 8 carbon atoms (“C1-8 alkoxy”), 1 to 6 carbon atoms (“C1-6alkoxy”), 1 to 4 carbon atoms (“C1-4 alkoxy”) or 1 to 3 carbon atoms(“C1-3 alkoxy”).

The term “halo” or “halogen” as used herein means fluorine, chlorine,bromine, or iodine.

The term “amino” as used herein means a moiety represented by thestructure NR₂, and includes primary amines, and secondary and tertiaryamines substituted by alkyl or aryl (i.e., alkylamino or arylamino,respectively). Thus, R₂ may represent two hydrogen atoms, two alkylmoieties, two aryl moieties, one aryl moiety and one alkyl moiety, onehydrogen atom and one alkyl moiety, or one hydrogen atom and one arylmoiety.

Alkyl (amino) is a moiety represented by the structure —RNR₂ andincludes an alkyl group as defined above attached to an amino group asdefined above, wherein the moiety is attached to another portion of amolecule via the alkyl group.

The term “cycloalkyl” means a non-aromatic, monocyclic or polycyclicring comprising carbon and hydrogen atoms.

The term “derivative” as used herein means a compound that is formedfrom a similar, beginning compound by attaching another molecule or atomto the beginning compound. Further, derivatives, according to theinvention, encompass one or more compounds formed from a precursorcompound through addition of one or more atoms or molecules or throughcombining two or more precursor compounds.

In one embodiment of the present invention, acetoin has beensuccessfully used as the precursor to make tetramethylpyrazine (TMP).Specifically, acetoin has successfully been reacted with ammoniumhydroxide and phosphoric acid (or diammonium phosphate) to yieldtetramethylpyrazine (TMP) in essentially quantitative yields (>80%yield) with an accompanying significant degree of purity. The reactionbetween acetoin and ammonium hydroxide produces almost exclusively TMPwith little to no detectable amounts of the molecules pyrazine andmethylpyrazine. As used herein, the terms “little to no detectableamounts”, “substantially no”, and “substantially zero” are used toindicate that the identified compound is present in an amount of lessthan 1.0% by weight, less than 0.5% by weight, or less than 0.1% byweight, based on the total weight of a reaction product.

Without being limited by theory, the different resulting pyrazinessynthesized from the reactions with sugar(s) versus acetoin as thecarbon source, surprisingly indicate that the carbon source dictates, toa significant degree, the distribution of pyrazines in reactions betweencarbon sources and nitrogen sources such as ammonium ions and/or aminoacids. In particular, it was surprisingly discovered that usingdifferent hydroxy ketones as the carbon source in the reaction to formpyrazines can produce an array of specific substituted pyrazines in acontrolled fashion.

In certain embodiments, at least one substituted pyrazine producedaccording to methods described herein is disubstituted. In someembodiments, at least one substituted pyrazine produced according tomethods described herein is trisubstituted. In various embodiments, atleast one substituted pyrazine produced according to methods describedherein is tetrasubstituted. In various embodiments, at least onesubstituted pyrazine produced according to methods of the presentinvention comprises at least one substituent group having 2 or morecarbon atoms. In certain embodiments, at least one substituted pyrazineproduced according to methods of the present invention comprises atleast one substituent group having 3 or more carbon atoms.

In some embodiments, at least one substituted pyrazine producedaccording to methods described herein is a branched pyrazine. As usedherein, the term “branched pyrazine” refers to the inclusion of alkylgroups on the pyrazine ring that are not linear in nature. For example,isobutyl, sec-butyl, and tert-butyl groups instead of the n-butyl group(this similarly applies to propyl and pentyl groups). As described inmore detail below, it was surprisingly discovered that by varying thealpha hydroxy ketone, the distribution of the pyrazines produced can bedictated. For example, use of acetoin in the reaction yields onlytetramethylpyrazine (about 99.5% or greater pure). Use of acetol in thereaction yields mostly dimethylsubstituted pyrazines (about 95% orgreater of the yield of pyrazines).

As illustrated in FIG. 1, for example, heated formulations comprisingnitrogen sources and at least one hydroxy ketone can produce pyrazines.Pyrazine rich solutions can be prepared in various ways. For example,one method can involve microwave heat treatment of a solution comprisingat least one amino acid and at least one hydroxy ketone. As illustratedat operation 100 of FIG. 1, for example, an aqueous reaction solutioncomprising at least one amino acid and at least one hydroxy ketone canbe formed. As illustrated at operation 104 of FIG. 1, for example, thereaction solution can be heated to a reaction temperature and held atthe reaction temperature for a reaction time which is sufficient toallow the reactions to undergo a reaction to form pyrazines. Asillustrated at operation 106 of FIG. 1, for example, pyrazines can thenbe optionally isolated from the reaction product using simpledistillation or other separation techniques known in the art.

In one embodiment, pyrazines are isolated from the reactant product byfirst using simple distillation to provide a distillate comprisingmostly water and pyrazines. This distillate can then be subjected toliquid liquid extraction with cyclohexane. The amount of cyclohexaneused in the liquid liquid extraction can be equal to about half theamount of the distillate used. For example, if 10 L of distillate isused, 5 L of cyclohexane can be used. The liquid liquid extraction ofthe distillate with cyclohexane can be repeated multiple times. In someembodiments, the liquid liquid extraction of the distillate withcyclohexane can be repeated at least 5 times. Following the liquidliquid extraction, the cyclohexane containing the extracted pyrazinescan be dried with any drying agent known in the art. For example, sodiumsulfate, magnesium hydroxide, and/or molecular sieves can be used to drythe cyclohexane. After drying, the pyrazines can be isolated (i.e., thecyclohexane can be removed) by simple distillation and/or rotaryevaporation.

As discussed above, using different hydroxy ketones as the carbon sourcein the reaction to form pyrazines can produce an array of specificsubstituted pyrazines. In certain embodiments, the carbon sourcecomprises acetoin. As discussed in more detail in Example 1 below, forexample, when acetoin serves as the sole carbon source in reactions witha nitrogen source (e.g., ammonium hydroxide (NH₄OH) and phosphoric acid(H₃PO₄), diammonium phosphate, etc.) to make pyrazines, the onlypyrazine produced is tetramethylpyrazine. Furthermore, branchedpyrazines, such as isopropylpyrazine, are not synthesized via theaddition of amino acids (such as, for example, leucine or free aminoacids from hydrolyzed F1 protein) to a reaction which contains acetoin,NH₄OH and H₃PO₄. Heating the reaction at higher temperatures or forlonger periods of time does not change the result (i.e., thattetramethylpyrazine (TMP) is the sole pyrazine synthesized fromacetoin). Hence, carbon sources other than acetoin can be synthesized ifpyrazines other than TMP are desired from this synthetic approach.

In some embodiments, the carbon source can comprise 1-hydroxyacetone(also referred to as 1-OH-acetone, acetol, or 1-hydroxy-2-propanone).Acetol, an alpha-hydroxy ketone, is the simplest hydroxy ketone. Asdiscussed in more detail below, acetol can be produced by thedegradation of various sugars. For example, it can be formed as anintermediate in a Maillard reaction (i.e., reaction of sugar(s) andamino acid(s) to form pyrazines), and then the acetol can react furtherto form other compounds. In some embodiments of the present invention,the carbon source can comprise 1-hydroxy-2-butanone. The structures of1-OH-acetone and 1-OH-2-butanone appear below. It is noted that for1-OH-acetone, a methyl group is bound to one side of the carbonyl andfor 1-OH-2-butanone, an ethyl group is bound to one side of thecarbonyl. Without being limited by theory, these structural features ofthe two α,β-hydroxyketones can dictate the structure of the resultingpyrazines.

As illustrated in Example 2 below, for example, reaction of1-hydroxyacetone as a carbon source and NH₄OH as a source of base andnitrogen can produce an array of pyrazines. When 1-hydroxyacetone(acetol) serves as the carbon source, an array of specific alkylsubstituted pyrazines can be produced. For example, exemplary pyrazinesprovided from the reaction of 1-hydroxyacetone with NH₄OH can include:2,6-dimethylpyrazine; 2,5-dimethylpyrazine; 2-ethyl-5-methylpyrazine;2-ethyl-6-methylpyrazine; 2,3,5-trimethylpyrazine;2-ethyl-3,5-dimethylpyrazine; 2-ethyl-2,5-dimethylpyrazine;2,3,5,6-tetramethylpyrazine; 2,3,5-trimethyl-6-ethylpyrazine;2,6-dimethyl-3-propylpyrazine; 2,5-diethyl-3,6-dimethylpyrazine;2,6-dimethyl-3-(2-methylbutyl)pyrazine;2,5-dimethyl-3-(2-methylbutyl)pyrazine;2,5-dimethyl-3-(3-methylbutyl)pyrazine; 2,5-dimethyl-3-propylpyrazine;2,5-dimethyl-3-cis-propenylpyrazine; 2-isopropenyl-3,6-dimethylpyrazine;2-(2-methylpropyl)-3,5-dimethylpyrazine;2,6-dimethyl-3-isobutylpyrazine;2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine;trimethylpyrazine; 2,5-dimethyl-3-ethylpyrazine; tetramethylpyrazine;2,3-diethyl-5-methylpyrazine; 2,5-dimethyl-3-propenylpyrazine;2,3,5-trimethyl-6-isopropylpyrazine; 2-acetyl-4,5-dimethylpyrazine; and3,5-dimethyl-2-methylpropylpyrazine.

When 1-hydroxy-2-butanone is used as the sole carbon source, pyrazineshaving ethyl groups attached can be produced. For example, asillustrated in Example 4 below, pyrazines synthesized from a reactionusing 1-OH-2 Butanone and NH4OH can include 2,6-diethylpyrazine;2,5-diethylpyrazine; 2-ethyl-3,5,6-trimethylpyrazine; 3,5-dimethyl)-2-(n-propyl)pyrazine; 3,6-dimethyl-2-(n-propyl) pyrazine;2,5-diethyl-3-methylpyrazine; 2,3-diethyl-5,6-dimethylpryazine;trans-3-methyl-2-(n-propyl)-6-(butenyl)pyrazine; and2,5-dimethyl-3-ethylpyrazine.

When 1-hydroxyacetone (acetol), 2-hydroxy-3-butanone (acetoin), and1-hydroxy-2-butanone are used as carbons sources in reactions withnitrogen sources, the pyrazine and methylpyrazine molecules do notappear in the array of structures and pyrazines produced. It is notedthat other hydroxy ketones can be used to produce alternative arrays ofpyrazine. Using different hydroxy ketones as the carbon source in areaction to produce pyrazines can not only minimize the formation ofpyrazine and methylpyrazine, but also allows one to tailor the reactionso that other desired substituted pyrazines are produced in a controlledfashion.

As illustrated in FIG. 1 at operation 102 for example, amino acidsand/or aldehydes can optionally be added to the reaction solutioncomprising at least one hydroxy ketone and a nitrogen source. Asillustrated in Example 3 below, for example, addition of amino acids orselected aldehyde(s) can increase not only the number of synthesizedpyrazines, but also can increase the yield of pyrazines. For example, incertain embodiments the reaction solution can comprise an amino acidselected from the group consisting of serine, alanine, leucine,isoleucine, valine, threonine, phenylalanine, and combinations thereof.Similarly, any alkyl aldehyde can be employed to increase the number ofsynthesized pyrazines as well as the yield of pyrazines. For example, incertain embodiments the reaction solution can comprise an alkyl aldehydeselected from the group consisting of acetaldehyde, propanal,isopropanol, butanal, isobutanal, sec butanal, and combinations thereof.

Increased reaction time and temperature can produce increased pyrazinesyield up to the point where a black tar substance is produced. Reactiontemperature can be about 30° C. or greater, about 90° C. or greater,about 100° C. or greater, about 120° C. or greater, or about 140° C. orgreater for example. In some embodiments, the reaction temperature canbe about 90° C. to about 150° C., or about 120° C. to about 140° C.Reaction time can be about 4 hours or greater, 8 hours or greater, about12 hours or greater, 16 hours or greater, or about 24 hours or greater,for example. In various embodiments, the reaction time can be about 4 toabout 24, or about 12 to about 20 hours. In certain embodiments, thereaction time can be about 16 hours.

In various embodiments, the molar ratio of the hydroxy ketone to thenitrogen source can affect the yield of pyrazines. The molar ratio ofhydroxy ketone:nitrogen source (e.g., NH₄OH) can be about 1:0.5, about1:1, about 1:2, or about 1:2.5, for example. In certain embodiments, theratio of hydroxy ketone to nitrogen source can be between about 1:0.5 toabout 1:2.5, or between about 1:1 to about 1:2. In certain embodiments,the ratio of hydroxy ketone to nitrogen source can be about 1:2.

Increasing the pH of the reaction solution can also result in increasedamounts of pyrazines. A favored pH range can be about 7.5 to about 10.5,or about 8.5 to about 9.5. In some embodiments, the pH of the reactionsolution can be about 8.0 or greater, about 8.5 or greater, about 9.0 orgreater or about 10.0 or greater. A small addition of NaOH or KOH, forexample, can be used to increase the pH of the reaction solution.

Selective Formation of Pyrazines Using Sugar(s) as the Carbon Source

In various embodiments of the present invention, the selective formationof substituted pyrazines has been optimized using at least one sugar(e.g., glucose, high fructose tobacco syrup (HFTS)) as the carbon sourceand ammonium ions, protein and/or amino acids as the nitrogen source. Asdiscussed above, various reaction pathways to pyrazine-rich formulationshave been previously used to make pyrazines using a sugar as a carbonsource. See, e.g., U.S. patent application Ser. No. 15/009,199 to Dubeet al., filed Jan. 28, 2016, which is herein incorporated by referencein its entirety. However, when sugars are used as the sole source in thereaction with a nitrogen source (e.g., ammonium hydroxide) to producepyrazines, the molecules pyrazine and methylpyrazine can be the dominantpyrazines produced. Even when free amino acids are employed asco-reagents, this trend is evident. It was surprisingly discovered thatby adjusting the pH of the carbon source before introducing it to thenitrogen source, substituted pyrazines can be selectively produced.

As illustrated in FIG. 2, for example, heated formulations comprisingamino acids and sugars can produce pyrazines. See, e.g., U.S. Pat. Pub.No. 2010/0037903 to Coleman III et al.; and Coleman III, On thesynthesis and characteristics of aqueous formulations rich in pyrazines,in Flavor Fragrance and Odor Analysis, Second Edition, Ray Marsili, ed.,Chapter 7, pp 135-182, CRC Press, Boca Raton, 2012; which are hereinincorporated by reference in their entireties. Pyrazine rich solutionscan be prepared in various ways. For example, one method can involvemicrowave heat treatment of a solution comprising at least one aminoacid and at least one sugar. As illustrated at operation 120 of FIG. 2,for example, an aqueous reaction solution comprising at least one aminoacid and at least one sugar can be formed. As illustrated at operation124 of FIG. 1, for example, the reaction solution can be heated to areaction temperature and held at the reaction temperature for a reactiontime which is sufficient to allow the reactions to undergo a reaction toform pyrazines.

As discussed above, acetol, a hydroxy ketone, can be produced by thedegradation of various sugars. Furthermore, when used as a carbon sourcein reactions with a nitrogen source, an array of substituted pyrazinescan be produced, which do not include the molecules pyrazine andmethylpyrazine. Accordingly, acetol can be a key intermediate in thereactions between sugar(s) and free amino acids and/or ammonium ions tomake pyrazines containing branched alkyl side chains.

Literature indicates that 1-hydroxy-2-propanone (acetol) can be producedfrom a C6 sugar such as glucose and a derivative of glucose, sorbitol.See, e.g., M. H. Mohamad, et. al., “A review of acetol: application andproduction”, Amer. J. Appl. Sci., 8, 1135-1139 (2011); M. A. Dasari,“Catalytic conversion of glycerol and sugar alcohols to value addedproducts”, Univ. Missouri-Columbia, ISBN-10, 0549727582, pp, 264; W.Yan, “Gas phase conversion of sugars to C3 chemicals, PhD Thesis,University of Missouri-Columbia, 2008; J. Hayami, Mechanism of acetolformation”, Bull. Chem. Soc. Japan, 34, 927-932(1961); P. F. Shaw, et.al., “Base catalyzed fructose degradation”, J. Agric. Food Chem., 16,979-982(1968); H. Weenen and W. Apeldoon, “Carbohydrate Cleavage in theMaillard Reaction”, Flavor Science, Recent Developments, A. Taylor andD. Mottran, eds., Royal Society of Chemistry, Special Publication #197,Cambridge, 1996; each of which are herein incorporated by reference intheir entireties. These references report on the conversion of sugars to1-hydroxy-2-propanone using phosphate buffers at elevated temperatures,a strong base such as NaOH at pH 11.5 while under reflux, Ni andPalladium catalysts under hydrogen pressure, and copper chromitecatalysts in the gas phase of heterogeneous reactions. Copper-chromitehas been considered to be the best catalyst. For most of the reactions,the conversion of substrates was greater than 91%. The yield of acetolwhen they used glycerol as carbon source was 32.2% at 220° C. Whensorbitol was used as a carbon source, the highest yield was 11.8% at280° C. and when glucose was used as a carbon source, the highest yieldof acetol was 8.99% at 280° C.

Cellulose has been converted to acetol with a 30% yield using a Sn basedcatalyst system. See, e.g., F. Chambon, et. al., Process fortransformation of lignocellulose biomass or cellulose by catalysts basedon Sn oxide and/or Sb oxide and a metal that is selected from Groups 8to 11, US Patent Application, US2013/028174A1, 2013, which is hereinincorporated by reference in its entirety.

Recently Novotny et al., (Czech J. Food Sci., 25, 119-130, 2007, whichis herein incorporated by reference) synthesized α-hydroxycarbonyl andα-dicarbonyl compounds via the degradation of monosaccharides. They usedthree different models comprised of an aqueous solution of potassiumperoxodisulfate, an alkaline solution of potassium peroxodisulfate, anda solution of sodium hydroxide, respectively. A total of sixα-hydroxycarbonyl and six α-dicarbonyl compounds were identified viaGC/MS. The maximum yield of α-dydroxycarbonyl (glycolaldehye, acetol,lactaldehyde, glyceraldehyde, 1,3-dihydroxyacetone and acetoin) whenglucose or fructose reacted with sodium hydroxide was approximately 4%.The yield was much lower in the aqueous solutions of potassiumperoxodisulfate (0.32%) and alkaline solution of potassiumperoxodisulfate (1.1%). Acetol and 1,3-dihydroxyacetone had the highestyield (2.52 and 1.02%, respectively) when sodium hydroxide was used.

As illustrated in Example 6 below, alpha hydroxy ketones (e.g., acetol)can be produced from sugar to ultimately be used as the carbon source insugar ammonia reactions. The acetol can be isolated via bothdistillation and column chromatography and then used as a carbon sourcein the reactions described herein. However, both techniques are timeconsuming (distillation) and expensive (chromatography). As such, it canbe preferable to avoiding having to isolate hydroxy ketones derived froma sugar source before using these hydroxy ketones in reactions with anitrogen source to produce pyrazines.

As illustrated in Example 7 below, for example, it has been surprisinglydiscovered that substituted pyrazines can be selectively formed from asugar carbon source without first isolating a hydroxy ketone (e.g.,acetol, acetoin, etc.) formed from the degradation of the sugar(s). Asillustrated at operation 110 of FIG. 2, for example, by treating thesugar(s) with a buffer before combining the sugar(s) with the nitrogensource, a different array of pyrazines can be produced than the array ofpyrazines produced from the reaction of sugar(s) that were notpre-treated with a buffer and a nitrogen source. The reactions can beoptimized such that the maximum amount of acetol and acetol-likecompounds are produced from the sugar carbon source. Without beinglimited by theory, the role of the buffer is to control the pH so thatthe maximum amount of hydroxy ketone(s) (e.g., acetol, acetoin, etc.)are produced from the degradation of the sugar.

In some embodiments, the buffer can be a sodium hydrogenphosphate/sodium hydroxide buffer with a pH of about 12. In variousembodiments, the buffer can include a potassium phosphate buffer with apH of about 6.5 to about 7.5. In certain embodiments, the buffer caninclude sodium carbonate, sodium sulfite, peroxodisulfate, sodiumphosphate, or combinations thereof. The selection of the buffer type,capacity, pH, and reaction temperature can affect the synthesis ofhydroxy ketones from the sugar carbon source, and can therefore impactthe array of pyrazines produced as well as the quantity of pyrazinesproduced from the subsequent reaction with a nitrogen source.

In various embodiments, the buffer can buffer at a pH of approximatelyneutral or in an alkaline range, such as at a pH greater than about 6,greater than about 8, or greater than about 10 (e.g., about 6 to about12). For example, in certain embodiments, the pH of the sugar carbonsource can be buffered to about 11 to about 12. In some embodiments, thepH of the sugar carbon source can be buffered to about 6.5 to about 7.5.

For example, glucose is a known carbon source for the production ofpyrazines. When employed as an intact molecule and reacted with ammoniumhydroxide, an array of pyrazines are produced, including the moleculespyrazine and methylpyrazine as the dominate pyrazines with much lesseramounts of dimethylpyrazines and significantly less amounts of highermolecular weight pyrazines. When the glucose is pre-reacted with NaOH atpH 12 followed by reaction with ammonium hydroxide, a very similar arrayof pyrazines are produced. However, it was surprisingly discovered thatif the glucose was treated with a potassium phosphate buffer at pH 6.5followed by reaction with ammonium hydroxide, only dimethylpyrazines andhigher molecular weight pyrazines were produced, thereby excluding theproduction of the less desirable molecules pyrazine and methylpyrazine.

Increased reaction time between the buffered sugar(s) carbon source andthe nitrogen source and temperature can produce increased pyrazinesyield up to the point where a black tar substance is produced. Reactiontemperature can be about 30° C. or greater, about 90° C. or greater,about 100° C. or greater, about 120° C. or greater, or about 140° C. orgreater for example. In some embodiments, the reaction temperature canbe about 90° C. to about 150° C., or about 120° C. to about 140° C.Reaction time can be about 30 mins or greater, about 60 mins or greater,about 90 mins or greater, or about 120 mins or greater, for example. Invarious embodiments, the reaction time can be about 30 mins to about 150mins, or about 60 mins to about 120 mins.

Increasing the pH of the reaction solution can also result in increasedamounts of pyrazines. A favored pH range can be about 7.5 to about 10.5,or about 8.5 to about 9.5. In some embodiments, the pH of the reactionsolution can be about 8.0 or greater, about 8.5 or greater, about 9.0 orgreater or about 10.0 or greater. A small addition of NaOH or KOH, forexample, can be used to increase the pH of the reaction solution.

In various embodiments of the present invention, as illustrated atoperation 122 of FIG. 2 for example, the addition of NH₄OH to the aminoacid/sugar reaction solution can increase the yield of pyrazines. TheNH₄OH/sugar molar ratio can have a dramatic influence on the yield ofpyrazines. For example, a molar ratio of sugar to NH₄OH of about 6:1 toabout 1:1, or about 5:1 to about 2:1 (e.g., about 5:1, about 2.5:1,about 2:1, or about 1.5:1), followed by heat treatment can produceformulations rich in pyrazines. In some embodiments, aqueous NH₄OH canslowly be added into the amino acid/sugar solution over the course ofthe reaction.

Different sugars and amino acids affect the types of pyrazines formed.See, e.g., Coleman and Steichen, 2006, Sugar and selected amino acidinfluences on the structure of pyrazines in microwave heat treatedformulations, J. Sci. Food Agric., 86, 380-391, which is hereinincorporated by reference in its entirety. For example, leucine andvaline produce more branched pyrazines with highly substituted subchainsand lower odor thresholds. Highly substituted pyrazines are relativelymore potent than pyrazines that are not as branched, and therefore canbe desirable in some applications. The substitution in the pyrazine canbe a result of the amino acid used in the reaction. Therefore, it can beadvantageous to select amino acids with branching, highly substitutedsubchains. With regard to sugars, rhamnose can be an ideal sugar forpyrazine formation, followed by fructose and then glucose.

As illustrated at operation 126 of FIG. 2, for example, following thereaction, optionally pyrazines can be isolated from the reaction productusing simple distillation, rotary evaporation, or other separationtechniques known in the art. In certain embodiments, rotary evaporationcan be a preferred isolation technique in a scaled up process ofderiving tobacco-derived pyrazines.

Uses of Substituted Pyrazines in Tobacco Products

As described above, pyrazines generated according to the presentinvention can be useful as components (e.g., flavorants) incorporatedinto tobacco products, for example. The tobacco product to which thematerials of the invention are added can vary, and can include anyproduct configured or adapted to deliver tobacco or some componentthereof to the user of the product. Exemplary tobacco products includesmoking articles (e.g., cigarettes), smokeless tobacco products, andaerosol-generating devices that contain a tobacco material or otherplant material that is not combusted during use.

In various embodiments of the present invention, pyrazines can beincorporated into smoking articles in the form of a flavorant in atobacco composition and/or in a filter element of a smoking article. Forexample, pyrazines can be incorporated into a top dressing or casing ofa tobacco product. Referring to FIG. 6, there is shown a smoking article10 in the form of a cigarette and possessing certain representativecomponents of a smoking article that can contain products derived fromthe cellulosic sugar materials of the present invention. The cigarette10 includes a generally cylindrical rod 12 of a charge or roll ofsmokable filler material (e.g., about 0.3 to about 1.0 g of smokablefiller material such as tobacco material) contained in a circumscribingwrapping material 16. The rod 12 is conventionally referred to as a“tobacco rod.” The ends of the tobacco rod 12 are open to expose thesmokable filler material. The cigarette 10 is shown as having oneoptional band 22 (e.g., a printed coating including a film-formingagent, such as starch, ethylcellulose, or sodium alginate) applied tothe wrapping material 16, and that band circumscribes the cigarette rodin a direction transverse to the longitudinal axis of the cigarette. Theband 22 can be printed on the inner surface of the wrapping material(i.e., facing the smokable filler material), or less preferably, on theouter surface of the wrapping material.

At one end of the tobacco rod 12 is the lighting end 18, and at themouth end 20 is positioned a filter element 26. The filter element 26positioned adjacent one end of the tobacco rod 12 such that the filterelement and tobacco rod are axially aligned in an end-to-endrelationship, preferably abutting one another. Filter element 26 mayhave a generally cylindrical shape, and the diameter thereof may beessentially equal to the diameter of the tobacco rod. The ends of thefilter element 26 permit the passage of air and smoke therethrough. Aplug wrap 28 enwraps the filter element and a tipping material (notshown) enwraps the plug wrap and a portion of the outer wrappingmaterial 16 of the rod 12, thereby securing the rod to the filterelement 26.

The filter element of the invention typically comprises multiplelongitudinally extending segments. Each segment can have varyingproperties and may include various materials capable of filtration oradsorption of particulate matter and/or vapor phase compounds.Typically, the filter element of the invention includes 2 to 6 segments,frequently 2 to 4 segments. In one preferred embodiment, the filterelement includes a mouth end segment, a tobacco end segment and acompartment therebetween. This filter arrangement is sometimes referredto as a “compartment filter” or a “plug/space/plug” filter. Thecompartment may be divided into two or more compartments as described ingreater detail below.

In various embodiments, the filter element can comprise an adsorbent inthe form of an activated carbon material, wherein the activated carbonis capable of removing at least one gas phase component of mainstreamsmoke is incorporated into the filter element. In certain embodiments,the filter element 26 can include ventilation holes 30 that extendthrough the tipping paper (not shown) and the plug wrap 28 and, thus,provide air dilution of mainstream smoke. The ventilation holes 30 maybe configured as a single line of perforations extendingcircumferentially around the filter element 26 or may comprise severallines of perforations. As would be understood, the exact count and sizeof the ventilation holes 30 will vary depending on the desired level ofair dilution.

In various embodiments of the present invention, pyrazines obtainedthrough methods disclosed herein can be incorporated into smokelesstobacco products in the form of a flavorant in a smokeless tobaccoformulation. The form of the smokeless tobacco product of the inventioncan vary. In one particular embodiment, the product is in the form of asnus-type product containing a particulate tobacco material and aflavorant comprising a pyrazine obtained through methods of the presentinvention. Manners and methods for formulating snus-type tobaccoformulations will be apparent to those skilled in the art of snustobacco product production. For example, as illustrated in FIG. 7, anexemplary pouched product 300 can comprise an outer water-permeablecontainer 320 in the form of a pouch which contains a particulatemixture 315 adapted for oral use. The orientation, size, and type ofouter water-permeable pouch and the type and nature of the compositionadapted for oral use that are illustrated herein are not construed aslimiting thereof.

In various embodiments, a moisture-permeable packet or pouch can act asa container for use of the composition within. Thecomposition/construction of such packets or pouches, such as thecontainer pouch 320 in the embodiment illustrated in FIG. 7, may bevaried as noted herein. For example, suitable packets, pouches orcontainers of the type used for the manufacture of smokeless tobaccoproducts, which can be modified according to the present invention, areavailable under the tradenames CatchDry, Ettan, General, Granit,Goteborgs Rape, Grovsnus White, Metropol Kaktus, Mocca Anis, Mocca Mint,Mocca Wintergreen, Kicks, Probe, Prince, Skruf and TreAnkrare. A pouchtype of product similar in shape and form to various embodiments of apouched product described herein is commercially available as ZONNIC(distributed by Niconovum AB). Additionally, pouch type productsgenerally similar in shape and form to various embodiments of a pouchedproduct are set forth as snuff bag compositions E-J in Example 1 of PCTWO 2007/104573 to Axelsson et al., which is incorporated herein byreference, which are produced using excipient ingredients and processingconditions that can be used to manufacture pouched products as describedherein.

The amount of material contained within each pouch may vary. In smallerembodiments, the dry weight of the material within each pouch is atleast about 50 mg to about 150 mg. For a larger embodiment, the dryweight of the material within each pouch preferably does not exceedabout 300 mg to about 500 mg.

In some embodiments, each pouch/container can have disposed therein aflavor agent member, as described in greater detail in U.S. Pat. No.7,861,728 to Holton, Jr. et al., which is incorporated herein byreference. The flavor agent member can comprise a flavorant comprising apyrazine derived via methods of the present invention, as discussedabove. If desired, other components can be contained within each pouch.For example, at least one flavored strip, piece or sheet of flavoredwater dispersible or water soluble material (e.g., a breath-fresheningedible film type of material) may be disposed within each pouch alongwith or without at least one capsule. Such strips or sheets may befolded or crumpled in order to be readily incorporated within the pouch.See, for example, the types of materials and technologies set forth inU.S. Pat. No. 6,887,307 to Scott et al. and U.S. Pat. No. 6,923,981 toLeung et al.; and The EFSA Journal (2004) 85, 1-32; which areincorporated herein by reference.

In various embodiments, the outer water-permeable pouch can comprise PLAor other pouch materials known in the art. Descriptions of variouscomponents of snus types of products and components thereof also are setforth in US Pat. App. Pub. No. 2004/0118422 to Lundin et al., which isincorporated herein by reference. See, also, for example, U.S. Pat. No.4,607,479 to Linden; U.S. Pat. No. 4,631,899 to Nielsen; U.S. Pat. No.5,346,734 to Wydick et al.; and U.S. Pat. No. 6,162,516 to Derr, and USPat. Pub. No. 2005/0061339 to Hansson et al.; each of which isincorporated herein by reference. See, also, the types of pouches setforth in U.S. Pat. No. 5,167,244 to Kjerstad, which is incorporatedherein by reference. Snus types of products can be manufactured usingequipment such as that available as SB 51-1/T, SBL 50 and SB 53-2/T fromMerz Verpackungmaschinen GmBH. Snus pouches can be provided asindividual pouches, or a plurality of pouches (e.g., 2, 4, 5, 10, 12,15, 20, 25 or 30 pouches) can connected or linked together (e.g., in anend-to-end manner) such that a single pouch or individual portion can bereadily removed for use from a one-piece strand or matrix of pouches.

The invention is not limited to snus-type smokeless tobacco products.For example, the mixture of tobacco material and flavorants comprisingat least one pyrazine derived via the methods described herein can alsobe incorporated into various smokeless tobacco forms such as loose moistsnuff, loose dry snuff, chewing tobacco, pelletized tobacco pieces,extruded tobacco strips or pieces, finely divided or milled agglomeratesof powdered pieces and components, flake-like pieces (e.g., that can beformed by agglomerating tobacco formulation components in a fluidizedbed), molded tobacco pieces (e.g., formed in the general shape of acoin, cylinder, bean, cube, or the like), pieces of tobacco-containinggum, products incorporating mixtures of edible material combined withtobacco pieces and/or tobacco extract, products incorporating tobacco(e.g., in the form of tobacco extract) carried by a solid inediblesubstrate, and the like. For example, the smokeless tobacco product canhave the form of compressed tobacco pellets, multi-layered extrudedpieces, extruded or formed rods or sticks, compositions having one typeof tobacco formulation surrounded by a different type of tobaccoformulation, rolls of tape-like films, readily water-dissolvable orwater-dispersible films or strips (see, for example, US Pat. Appl. Pub.No. 2006/0198873 to Chan et al.), or capsule-like materials possessingan outer shell (e.g., a pliable or hard outer shell that can be clear,colorless, translucent or highly colored in nature) and an inner regionpossessing tobacco or tobacco flavor (e.g., a Newtonian fluid or athixotropic fluid incorporating tobacco of some form).

In some embodiments, smokeless tobacco products of the invention canhave the form of a lozenge, tablet, microtab, or other tablet-typeproduct. See, for example, the types of lozenge formulations andtechniques for formulating or manufacturing lozenges set forth in U.S.Pat. No. 4,967,773 to Shaw; U.S. Pat. No. 5,110,605 to Acharya; U.S.Pat. No. 5,733,574 to Dam; U.S. Pat. No. 6,280,761 to Santus; U.S. Pat.No. 6,676,959 to Andersson et al.; U.S. Pat. No. 6,248,760 toWilhelmsen; and U.S. Pat. No. 7,374,779; US Pat. Pub. Nos. 2001/0016593to Wilhelmsen; 2004/0101543 to Liu et al.; 2006/0120974 to Mcneight;2008/0020050 to Chau et al.; 2009/0081291 to Gin et al.; and2010/0004294 to Axelsson et al.; which are incorporated herein byreference.

Depending on the type of smokeless tobacco product being processed, thetobacco product can include one or more additional components inaddition to the tobacco material and the flavorants comprising at leastone pyrazine derived from methods of the present invention. For example,the tobacco material and the tobacco-derived flavorants can beprocessed, blended, formulated, combined and/or mixed with othermaterials or ingredients, such as other tobacco materials or flavorants,fillers, binders, pH adjusters, buffering agents, salts, sweeteners,colorants, disintegration aids, humectants, and preservatives (any ofwhich may be an encapsulated ingredient). See, for example, thoserepresentative components, combination of components, relative amountsof those components and ingredients relative to tobacco, and manners andmethods for employing those components, set forth in US Pat. Pub. Nos.2011/0315154 to Mua et al. and 2007/0062549 to Holton, Jr. et al. andU.S. Pat. No. 7,861,728 to Holton, Jr. et al., each of which isincorporated herein by reference.

In various embodiments, at least one pyrazine derived from methodsdescribed herein can be incorporated into smokeless tobacco products inthe form of a flavorant in an electronic smoking article. There havebeen proposed numerous smoking products, flavor generators, andmedicinal inhalers that utilize electrical energy to vaporize or heat avolatile material, or attempt to provide the sensations of cigarette,cigar, or pipe smoking without burning tobacco to a significant degree.See, for example, the various alternative smoking articles, aerosoldelivery devices and heat generating sources set forth in the backgroundart described in U.S. Pat. No. 7,726,320 to Robinson et al., U.S. Pat.Pub. Nos. 2013/0255702 to Griffith Jr. et al., 2014/0000638 to Sebastianet al., 2014/0060554 to Collett et al., 2014/0096781 to Sears et al.,2014/0096782 to Ampolini et al., and 2015/0059780 to Davis et al., whichare incorporated herein by reference in their entirety.

An exemplary embodiment of an electronic smoking article 200 is shown inFIG. 8. As illustrated therein, a control body 202 can be formed of acontrol body shell 201 that can include a control component 206, a flowsensor 208, a battery 210, and an LED 212. A cartridge 204 can be formedof a cartridge shell 203 enclosing a reservoir housing 244 that is influid communication with a liquid transport element 236 adapted to wickor otherwise transport an aerosol precursor composition stored in thereservoir housing to a heater 234. An opening 228 may be present in thecartridge shell 203 to allow for egress of formed aerosol from thecartridge 204. Such components are representative of the components thatmay be present in a cartridge and are not intended to limit the scope ofcartridge components that are encompassed by the present disclosure. Thecartridge 204 may be adapted to engage the control body 202 through apress-fit engagement between the control body projection 224 and thecartridge receptacle 240. Such engagement can facilitate a stableconnection between the control body 202 and the cartridge 204 as well asestablish an electrical connection between the battery 210 and controlcomponent 206 in the control body and the heater 234 in the cartridge.The cartridge 204 also may include one or more electronic components250, which may include an IC, a memory component, a sensor, or the like.The electronic component 250 may be adapted to communicate with thecontrol component 206. The various components of an electronic smokingdevice according to the present disclosure can be chosen from componentsdescribed in the art and commercially available.

In various embodiments, the aerosol precursor composition can comprise aflavorant comprising at least one pyrazine derived according to methodsof the present invention. Exemplary formulations for aerosol precursormaterials that may be used according to the present disclosure aredescribed in U.S. Pat. No. 7,217,320 to Robinson et al.; U.S. Pat. Pub.Nos. 2013/0008457 to Zheng et al.; 2013/0213417 to Chong et al.;2014/0060554 to Collett et al.; and 2014/0000638 to Sebastian et al.,the disclosures of which are incorporated herein by reference in theirentirety. Other aerosol precursors that can incorporate thetobacco-derived pyrazines described herein include the aerosolprecursors that have been incorporated in the VUSE® product by R. J.Reynolds Vapor Company, the BLU™ product by Imperial Tobacco, the MISTICMENTHOL product by Mistic Ecigs, and the VYPE product by CN CreativeLtd. Also desirable are the so-called “smoke juices” for electroniccigarettes that have been available from Johnson Creek Enterprises LLC.

EXPERIMENTAL

Aspects of the present invention are more fully illustrated by thefollowing examples, which are set forth to illustrate certain aspects ofthe present invention and are not to be construed as limiting thereof.

Example 1

Pyrazines are produced using acetoin (3-hydroxy-2-butanone) instead ofsugar(s) as the carbon source in reactions with ammonia.

Acetoin, ammonium hydroxide (28-30%), leucine, dichloromethane, andphosphoric acid (H₃PO₄) are obtained from Sigma-Aldrich (St. Louis,Mo.). F1 protein is obtained from R. J. Reynolds Tobacco Co.(Winston-Salem, N.C.) and hydrolyzed. The weight percent of hydrolyzedamino acids in all of the hydrolyzed solutions is in the range of50-55%. All pyrazine synthesis reactions are performed in a 40 mL Parrvessel. In each reaction, 0.8 gram of acetoin is mixed with 1.8 mL ofNH₄OH and 0.6 mL of H₃PO₄, and then enough hydrolyzed F1 protein (20 mL)is added to make the mass of amino acids equal to 0.4 gram. For example,when 40 grams of F1 protein is hydrolyzed in 1 liter solution, theweight percent of amino acids in the solution is equal to 50%, which isequal to 20 grams of amino acids in 1 liter solution. In order to use0.4 gram of amino acids in a reaction, only 20 mL of above solution isadded to the reaction vessel. In some of the reactions, instead ofhydrolyzed F1 protein, leucine is used as a source of amino acid andonly 20 mL of water is added to adjust the volume. For all solutions,the pH is adjusted to 8.0 and then reaction is started. After completionof each reaction, the mixture is extracted with 30 mL of dichloromethane(DCM). Next, 200 μL of the DCM extract is diluted to 1 mL using DCM andanalyzed via GC/MS.

All GC/MS analyses are performed using a 6890 GC equipped with a 5973Mass Selective detector (MSD) from Agilent (Wilmington, Del.).Separations are obtained using a DB-WAXTER capillary column (30 mlong×250 μm I.D. with a film thickness of 0.25 μm) from J&W (Wilmington,Del.). The following operating parameters are used for each analysis:

Injection Port Temp 260° C. Purge Valve 3 mL/min Purge Time 1 min TotalFlow 24 mL/min Constant Flow 1 mL/min Injection Volume 2 μL, split 1:20Column Oven Initial Temp 50° C. Column Oven Initial Time 3 min ColumnOven Ramp Rate 15° C./min Column Oven Final Temp 250° C. Column OvenFinal Time 1 min MSD Transfer line Temp 260° C.MS Wiley library is used to identify each pyrazine.

In a first reaction, acetoin (0.8 gram) is reacted with NH₄OH (1.8 mL)and H₃PO₄ (0.6 mL) at pH=8 at 90° C. for 12-15 hours. More than 90% ofacetoin is converted to tetramethylpyrazine (TMP). Next, the reaction isrepeated using identical conditions, but instead of heating the reactionat 90° C. for 12-15 hours, it is heated only for 4 hours at 120° C.Results are similar to those obtained at 90° C. and 12-15 hours.

Next, in order to determine if branched pyrazines can be synthesized, anamino acid (leucine) is added to the reaction reagent mixture. For thispurpose, 0.8 gram acetoin+1.8 mL of NH₄OH+0.6 mL of H₃PO₄ and 0.25 gramof leucine are mixed with 20 mL of H₂O and pH is adjusted to 8. Then thereaction is heated for 18 hours at 120° C. and then the reaction mixtureis extracted with 30 mL of DCM and analyzed via GC/MS. Only TMP(t_(R)=8.2 min) and some acetoin (t_(R)=6.1 min) are detected. Nobranched pyrazines are detected.

Next, a similar reaction is performed, but instead of leucine and H₂O,20 mL of hydrolyzed F1 protein is used for the reaction. The reaction isperformed in the Parr vessel at 120° C. for 18 hours. After cooling thereactant, 30 mL DCM is used to extract the pyrazines. Again, no branchedpyrazines were observed, only TMP.

In order to determine whether ammonia is consuming all the acetoin andthus preventing the amino acid(s) from reacting with acetoin, anothersource of base (NaOH) is used instead of NH₄OH to assure that basic,pH>8, reaction conditions are maintained. For this purpose, tworeactions are performed. In the first reaction, 0.8 acetoin is mixedwith 0.25 gram of leucine and 20 mL of 0.1N NaOH (pH=12), while in thesecond reaction the pH is adjusted to 8.2 using H₃PO₄. Both reactionsare heated at 120° C. for 8 hours using the Parr vessel. After coolingthe reaction, the reactants are extracted with DCM and analyzed viaGC/MS. No pyrazines, not even TMP, are detected.

The above reactions illustrate that the addition of amino acids to thereaction of acetoin and NH₄OH does not produce branched pyrazines.Furthermore, it is discovered that acetoin is not a key intermediate inthe reactions between sugar(s) and free amino acids to make pyrazinescontaining branched alkyl side chains.

Example 2

Hydroxy ketone carbon sources other than acetoin are utilized toselectively produce pyrazines other than TMP.

1-OH-acetone, 1-OH-2-butanone, ammonium hydroxide (28-30%), phosphoricacid (H₃PO₄), isoleucine, threonine, and isovaleraldehyde are obtainedfrom Sigma-Aldrich (St. Louis, Mo.). F1 proteins derived from a plant ofthe Nicotiana species are obtained from R. J. Reynolds Tobacco Co.(Winston-Salem, N.C.) and hydrolyzed to form amino acids. The weightpercent of hydrolyzed amino acids in all of the hydrolyzed solutions arein the range of 50-55%.

All pyrazine synthesis reactions are performed in a 40 mL Parr vessel.In each reaction, 1 gram of 1-OH-acetone or 1-OH-2-butanone is mixedwith 0.25, 0.5, 1 and 1.25 mL of NH₄OH and 10 mL of H₂O. Each reactionis mixed and heated at different temperatures (100-140° C.) for a periodof 4-24 hours. The pH levels for most of the reactions are approximately11 (no adjustments are made). However, in the reactions in which the pHlevel is adjusted to 8, concentrated H₃PO₄ is used to lower the pH.

After the completion of each reaction, the mixture is extracted with20-25 mL of dichloromethane. In each extraction, 250 μg of deuterated2-methylpyrazine is used as an internal standard for allquantifications. For all reactions, the mixtures are stirred using amagnetic stirrer during the reaction process.

All GC/MS analyses are performed using the same equipment and operatingparameters as those used in Example 1 above. MS Wiley library is used toidentify each pyrazine. For quantitative analysis, pyrazines arequantified using single ion monitoring mode. Each pyrazine is quantifiedagainst the mass of internal standard (250 μg) added to the extractionsolvent.

Initially, 1-hydroxyacetone is reacted with NH₄OH at different ratios,temperatures, pH levels, and reaction times to maximize the percentyield of pyrazines. The pyrazines detected from the reaction of1-OH-acetone with NH₄OH include: 2,6-dimethylpyrazine;2,5-dimethylpyrazine; 2-ethyl-5-methylpyrazine;2-ethyl-6-methylpyrazine; 2,3,5-trimethylpyrazine;2-ethyl-3,5-dimethylpyrazine; 2-ethyl-2,5-dimethylpyrazine;2,3,5,6-tetramethylpyrazine; 2,3,5-trimethyl-6-ethylpyrazine;2,6-dimethyl-3-propylpyrazine; 2,5-diethyl-3,6-dimethylpyrazine;2,6-dimethyl-3-(2-MethylButyl)pyrazine;2,5-dimethyl-3-(2-MethylButyl)pyrazine;2,5-dimethyl-3-(3-MethylButyl)pyrazine; 2,5-dimethyl-3-propylpyrazine;2,5-dimethyl-3-cis-propenylpyrazine; 2-isopropenyl-3,6-dimethylpyrazine;2-(2-methylpropyl)-3,5-dimethylpyrazine;2,6-dimethyl-3-isobutylpyrazine;2-(2-methylpropyl)-3,5,6-trimethylpyrazine.

In a first part of this study, reactions are performed at two differentpH levels (8.0 and 11.0) to determine which pH levels provide thehighest yield and greatest number of pyrazines. In a first reaction, 1mL of 1-OH-acetone is reacted with 0.5 mL of NH₄OH and 10 mL of H₂O. ThepH level for this reaction is measured to be around 11.0. In a secondreaction, the same volume of reactant is mixed, but the pH level isadjusted to 8.0 using concentrated H₃PO₄. Both reactions are heated at120° C. for 12 hours. The percent yield of the pyrazines is higher whenthe pH level is around 11.0. For this purpose, the pH level of laterexperiments is not adjusted and reactions are performed at pH 11 orhigher.

The effects of temperature (100, 110, 120, 130 and 140° C.) on thesynthesis of pyrazines using 1-OH-acetone (1 g) and NH₄OH (1 g) with a1:2 mole ratio of C:N in 10 mL of H₂O (reaction time 12 hours) is textednext. Pyrazine yield increases as the temperature increases, stillshowing an increase in pyrazine yield as the temperature reached 140° C.

The effect of varying reaction times (4, 8, 12, 16, and 24 hours) on thesynthesis of pyrazines using 1-OH-acetone (1 g) and NH₄OH (1 g) with a1:2 mole ratio of C:N in 10 mL of H₂O is tested. Maximum yields ofpyrazines are obtained when the reaction time is 16 hours.

The effect of varying the 1-OH-acetone:NH₄OH molar ratio (1:0.5, 1:1,1:2, and 1:2.5, 1-OH-acetone and NH₄OH) reaction in 10 mL of H₂O on theyield of pyrazines at 120° C. after 12 hours is tested. The optimumratio is 1:2, which is equal to 1 gram of 1-OH-acetone and 1 mL ofNH₄OH. When a higher volume of NH₄OH is used in the reaction, the yieldof the reaction drops by more than 10%.

In summary, reaction conditions (temperature, time, C:N ratio, and pH)are optimized to maximize quantity of pyrazines. Results demonstratedthat at optimized conditions (C:N=1:2, temperature=120° C., reactiontime=16 hours, and pH=11-12) at least 19-20 different pyrazines aresynthesized using hydroxyacetone as the sole carbon source. The absenceof any detectable amounts of the molecules pyrazine and/ormethylpyrazine from the synthesized pyrazines supports the discoverythat the carbon source (i.e., the α,β-hydroxyketone) influences thestructures of the pyrazines produced from reaction of the carbon sourcewith a nitrogen source.

Example 3

The effects of amino acids and aldehyde additions into a separatereaction of 1-OH-acetone and NH₄OH, according to the parameters ofExample 2 above, with C:N ratio of 1:2 mixed with 10 mL H₂O at 120° C.for 12 hours are measured.

Two different amino acids are tested as an additional source ofnitrogen. In each reaction, 0.2 grams of amino acid are added separatelyto each reaction so that it is possible to study how the additionalamino acids affect the pyrazine synthesis and its yield. In a separatereaction, isovaleraldehyde is added to the optimized reaction to studythe effect on pyrazine synthesis and yield. When the hydrolyzed F1protein is used as a source of additional nitrogen, 10 mL of hydrolyzedF1 proteins (which contain approximately 0.2 gram of different aminoacids) was used. In this reaction, H₂O is not added since the hydrolyzedF1 proteins are contained within the 10 mL of H₂O.

It is observed that when isoleucine is used as a possible additionalsource of nitrogen, the concentrations of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl) pyrazine increase. In aseparate reaction, when threonine is used as an additional source ofnitrogen, the concentrations of 2,5-dimethyl-3-(2-methylbutyl) pyrazineand 2,5-dimethyl-3-(3-methylbutyl) pyrazine increase and the2,6-dimethyl-3-(2-methylbutyl) pyrazine yield increases. It isinteresting to note that the total yields of pyrazines are similar whenthreonine or isoleucine is added to the reaction. Both compoundsincrease the total yield of pyrazines by more than 7%.

The addition of isovaleraldehyde to a separate reaction of 1-OH-acetoneand NH₄OH causes the percent yield of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl) pyrazine to increase from 0to 14 and 13 mg, respectively. The total yield of pyrazines increases bymore than 20 percent.

Instead of a pure amino acid, a mixture of amino acids prepared from thehydrolysis of F1 proteins is used in the reaction. Since hydrolyzed F1proteins are already in an aqueous solution, no water is added to themixture. For this purpose, 10 mL of hydrolyzed F1 proteins whichcontained about 0.2 g of amino acid and 10 mL of H₂O is reacted with-1-OH-acetone and NH₄OH with a C:N ratio of 1:2 at 120° C. for 16 hours.The yield of 2,5-dimethylpyrazine increases by more than 80% and theyield of 2,5-dimethyl-3-(2-methylbutyl) pyrazine and2,5-dimethyl-3-(3-methylbutyl) pyrazine increases from 0 to more than 1mg. Without being limited by theory, the alkyl portion of the amino acidis converted to a Strecker aldehyde which reacts with the ammoniumhydroxide to form an imine which in turn is incorporated into a pyrazinestructure.

The effects of different temperatures and C:N ratios (1:1 and 1:2) onthe synthesis of pyrazines using 1-OH-acetone and NH₄OH in the presenceof additional amino acids/aldehyde is tested. In these studies,increasing the temperature from 100 to 120° C. and C:N ratio, increasesthe yield of pyrazines.

In summary, addition of amino acids, selected aldehydes, or hydrolyzedF1 protein increases not only percent yield of certain pyrazines, butalso increases the number of synthesized pyrazines.

Example 4

Pyrazines are synthesized according to Examples 2 and 3 above, except1-OH-2-butanone is used as a carbon source instead of 1-OH-acetone.

For this purpose, 1 gram of 1-OH-2-butanone is reacted with 1 mL ofNH₄OH and 10 mL of H₂O at 120° C. for 16 hours. No methyl pyrazines areformed. All pyrazines formed contain ethyl or higher branched alkanes.The yield of pyrazines is, however, not as high as when 1-OH-acetone isused. The pyrazines synthesized from a reaction using 1-OH-2-butanoneand NH₄OH with C:N of 1:2 at 120° C. for 16 hours includes2,6-diethylpyrazine; 2,5-diethylpyrazine;2-ethyl-3,5,6-trimethylpyrazine; 3,5-dimethyl-2(n-propyl)pyrazine;3,6-dimethyl-2-(n-propyl)pyrazine; 2,5-diethyl-3-methylpyrazine;2,3-diethyl-5,6-dimethylpryazine;trans-3-methyl-2-(n-propyl)-6-(butenyl)pyrazine;2,5-dimethyl-3-ethylpyrazine.

As mentioned earlier, with 1-OH-acetone as the carbon source, themolecules pyrazine and methylpyrazine are not produced. With1-OH-2-butanone, the molecules pyrazine, methylpyrazine, anddimethylpyrazine are not produced, confirming that the carbon source isoverwhelmingly dictating the structure of the pyrazines. Results furthershow that by changing the carbon source from 1-OH-acetone to1-OH-2-butanone, one can control the type of pyrazines beingsynthesized.

Example 5

The reaction of 1-OH-acetone and NH₄OH according to Example 2 above isaccomplished on a larger scale using a larger Parr reactor with greaterreaction volume.

100 grams of 1-OH-acetone is reacted with 100 mL of NH₄OH and 1000 mL ofH₂O at 120° C. for 16 hours in a 1.5 liter Parr high pressure vessel.After the reaction is complete, the mixture is cooled and transferredinto a glass bottle.

It is noted that in the bottom of the vessel is a considerable amount ofa tar like material which is only soluble in MeOH. The addition of H₂Oto the top of this material causes it to become hard. It is discoveredthat concentration can play a significant role in the presence orabsence of the tar like material. For all optimization studies, theamount of tar at the bottom of reaction vessel is low. For this reason,a small volume of methanol (1 mL) is sufficient to dissolve everythingand include it with the remaining reaction material. For largereactions, the mass of tar is higher and at least 100-200 mL of methanolis required to dissolve it.

After the completion of the reaction, the aqueous solution is distilled(3×375 mL) at 130-140° C. In each distillation (375 mL), approximately175 mL of aqueous solution containing different pyrazines is collected(light yellow color—total volume about 500 mL). Next, the distilledmaterials (3×175 mL) are combined and passed through a C₁₈ column(15×2.5 cm packed with SPE material) in order to remove the pyrazinesfrom the water. After removal of the water from the C₁₈ column, trappedpyrazines are eluted using ethanol. Next, ethanol is removed using arotary evaporation and vacuum. Due to the presence of some water in thefinal product, pyrazines are extracted into MTBE and dried over sodiumsulfate. Next, MTBE is removed using a rotary evaporator and vacuum. Avial with the solution 1 label contains most of the pyrazines after MTBEis removed.

It is important to note here that three pyrazines are not distilled andremain in the reaction product due to their higher boiling points. Thiscontributed to a lower percent yield. These pyrazines are identified as2-(2-methylpropyl) 3,5-dimethylpyrazine (12.57 min),2,6-dimethyl-3-isobutylpyrazine (12.74 min), and 2(2-methylpropyl)3,5,6-trimethylpyrazine (12.95 min).

DCM (200-250 mL) is used to extract the remaining three pyrazines fromthe reaction solution after distillation. Next, DCM is removed from thesolution using a rotary evaporator and vacuum while the thick darksolution is later transferred to the second vial and labeled as solution2. FIG. 3 shows a chromatogram of this sample.

The yield of total pyrazines from distillation of 1200 mL of reactionsolution (100 grams of 1-OH-acetone+100 mL of NH₄OH) is about 60%compared to the 12 mL reaction (1 gram of 1-OH-acetone+1 mL of NH₄OH).This yield does not include the three pyrazines that remained in thepost distillation reaction mixture.

Solution 1 from above is used for the gas chromatography olfactometry(GCO) evaluations. The pyrazine sample is analyzed using an Agilent7890A Series GC with 5975C MSD and ODP3 equipped with a GerstelMultipurpose Sampler with SPME capability. The instrument method isdeveloped for the sample to obtain better separation of the pyrazinesand suitable analyzing duration for the olfactory analysts. The transferline in ODP3 is heated to 260° C. The sample is prepared by pipettingtwo drops of the sample into a 20 mL SPME screw cap vial. An empty vialis analyzed before and after the sample. The gas chromatographic columnseparated the pyrazines and they were detected/evaluated by humans usinga subjective smell test as they exited the column. The pyrazinesidentified include: methylpyrazine; 2,6-dimethylpyrazine;2,5-dimethylpyrazine; 2-ethyl-5-methylpyrazine;2-ethyl-6-methylpyrazine; trimethylpyrazine;2,5-dimethyl-3-propylpyrazine; 3-ethyl-2,5-dimethylpyrazine;2,5-dimethyl-3-isopropylpyrazine; 2-ethyl-3,5-dimethylpyrazine;tetramethylpyrazine; 2-methyl-5-propylpyrazine;2,3,5-trimethyl-6-propylpyrazine isomer 1; 2,3-diethyl-5-methylpyrazine;2,3,5-trimethyl-6-ethylpyrazine; 3,5-dimethyl-2-propylpyrazine;2,3,5-trimethyl-6-propylpyrazine isomer 2;2,3,5-trimethyl-6-propylpyrazine isomer 3; trimethyl-1-propenylpyrazine(Z)-isomer 1; 5H-clyclopentapyrazine, 6,7-dihydro-2,5-dimethyl pyrazineisomer 1; 5H-clyclopentapyrazine, 6,7-dihydro-2,5-dimethyl pyrazineisomer 2; trimethyl-1-propenylpyrazine (Z)-isomer 2;2,3-dimethyl-3-(1-propenyl)pyrazine (Z) isomer 1;trimethyl-1-propenylpyrazine (Z)-isomer 3;2,3-dimethyl-3-(1-propenyl)pyrazine (Z);2-isopropenyl-3,6-dimethylpyrazine; trimethyl-1-propenylpyrazine(Z)-isomer 4; trimethyl-1-propenylpyrazine (Z)-isomer 5;trimethyl-1-propenylpyrazine (Z)-isomer 6; trimethyl-1-propenylpyrazine(E)-isomer 1; trimethyl-2-propenylpyrazine; trimethyl-1-propenylpyrazine(E)-isomer 2. It is noted that the methylpyrazine compound, althoughpresent, was present in a lower amount than typical for traditionalsugar carbon source reactions.

Four flavor analysts evaluate the individual olfactory characters fromthe ODP portal in four separate sessions. The individual pyrazines,based on the four independent assessments, are very positive. Expecteddescriptors for the aroma of substituted pyrazines are found. Nutty,roasted, toasted, chocolate, peanut, musty, brown and complex arefrequent descriptors. These are all positive flavor characteristics.

Example 6

Alpha hydroxy ketones (acetol) are produced from sugar to ultimately beused as the carbon source in sugar ammonia reactions. Sodium hydroxideis used as a base to optimize different parameters such as sugar type,temperature, reaction time, pH, and base concentration in order tomaximize the yield of acetol.

Glucose, Fructose, 1-OH-acetone (acetol), 1-OH-2-butanone (acetoin),sodium hydroxide, sodium chloride, sodium sulfate anhydrous,hydrochloric acid, methanol, and dichloromethane are obtained fromSigma-Aldrich (St. Louis, Mo.). Synthesis reactions are performed in a40 mL Parr vessel or an open round bottle flask. For all reactions inwhich pH is controlled, reactions are performed in round bottom flasksunder reflux at 100° C. In each reaction, 0.25, 0.5, or 1.0 gram of adifferent sugar (glucose, fructose or mixture of both) is mixed with 25mL of 0.025, 0.05, 0.1 and 0.2 M NaOH. Each reaction is stirred andheated at different temperatures (90-140° C.) for a period of 1-12hours. The initial pH levels for most of the reactions are approximately12 and no adjustments are made during the reaction. However, for thereactions in which the pH level is adjusted to 9, hydrochloric acid isused. For reactions in which the pH is kept constant at 12 during theprocess, 10-40 μL of 10 M NaOH is used. After the completion of eachreaction, the mixture is cooled and the pH is adjusted to 6.0-6.5 using1 M HCl. Next, 1 mg of acetoin is added to the reaction mixture as aninternal standard. The mixture is extracted 4 times with 8-10 mL ofdichloromethane. All four extraction solvents are combined and driedwith sodium sulfate.

All GC/MS analyses are performed using the same equipment and operatingparameters as those used in Example 1 above. MS Wiley library is used toidentify peaks. For quantitative analysis, the calibration curve isprepared using a concentration of acetol and a response factor ratio ofacetol/acetoin. It is important to note here that acetoin is notdetected in any reactions performed at 90, 100 and 120° C. However,acetoin residue is found in the reaction where the temperature is set to140° C. For this purpose, acetoin concentrations are determined in thereaction and this value is accounted for in all calculations.

GC/FID is used to prepare the calibration curve for the quantificationof acetol in all reactions. Next, the following parameters are varied todetermine the optimum conditions for the synthesis of acetol.

First, two different type of sugars (glucose, fructose, and a mixture ofboth) are used to determine which sugar provides a higher yield ofacetol. In each reaction, 0.5 grams of sugar is mixed with 25 mL of0.05M NaOH. Each reaction is heated for 60 minutes at 100° C. Table 1below shows the reaction conditions and the corresponding mg of acetolthat were obtained from each reaction. It can be observed that glucosegenerated a high mass of acetol when it was used in the reaction.

TABLE 1 Effect of sugar type on synthesis of acetol % Yield wt. Temp,Time, mg base-mole Sugar Wt. gr pH NaOH ° C. min acetol* base Glucose(Glu) 0.5 12-7.5 0.05M 100 60 3.77 0.75-1.8 Fructose (Fru) 0.5 12-7.50.05M 100 60 2.97 0.59-1.4 Glu + Fru 0.25 + 0.25 12-7.5 0.05M 100 603.18 0.64-1.5 *% RSD varied from 2-5

In this part of study, since it is required to adjust the pH during thereaction process, all reactions are performed under reflux in an openround flask instead of in a closed Parr reactor. Three differentexperiments are performed. In the first experiment, the pH is measuredevery 10 minutes and no adjustment is made to the pH. In the secondexperiment, the pH is measured every 10 minutes and, if it is required,the pH is adjusted to the initial value (12.0) using 10M NaOH. In thethird experiment, initial pH is adjusted to 9.0 and then the reaction isstarted, with pH measurements taking place every 10 minutes (withoutadjustments).

Results from the pH measurements show that the pH drops from 12.0 toaround 9 within the first 10 minutes of the reaction at 100° C. At theend of 30 minutes, the pH of the reaction is approximately around 7-8and remains constant during the remaining reaction time. For theexperiments where the pH is maintained around 12.0, it becomes necessaryto add approximately 15-20 μL of 10M NaOH to the reaction every 10minutes. After 40 minutes, the pH remains constant around 11-12.

Table 2 below shows reaction conditions and the mg of acetol obtainedfrom each reaction using different pH conditions. When the pH is keptconstant around 12, the highest mass of acetol is obtained.

TABLE 2 Effect of pH on synthesis of acetol % Yield wt. Wt. Temp, Time,mg base-mole Sugar gr pH NaOH ° C. min acetol* base Glu 0.5  12-7.50.05M 100 60 2.79 0.56-1.4 Glu 0.5 12.0-12.0 0.05M 100 60 4.94 0.99-2.4Glu 0.5  9-7.5 0.05M 100 60 0.00 0.00-0.0 *% RSD varied from 5-9

Three different sugar concentrations are tested in order to determinehow sugar concentration effects acetol production. Therefore, reactionsare performed using 0.25, 0.5 and 1 gram of glucose in 25 mL of 0.05MNaOH solution. Each reaction is heated at 100° C. for 60 minutes using aParr reactor. Table 3 below shows the results of this study. It isobserved that when 0.25 gram of glucose is used in a reaction, thehighest amount of acetol is obtained.

Without being limited by theory, it is believed that when sugarconcentration is high, acid formation during the reaction processminimizes the production of acetol. When the sugar concentration isless, acid formation will take longer while the pH is high enough tocause the formation of acetol.

TABLE 3 Effect of Sugar Concentration on synthesis of acetol % Yield wt.Wt. Temp, Time, mg base-mole Sugar gr pH NaOH ° C. min acetol* base Glu0.25 12-7.5 0.05M 100 60 3.88 1.55-3.8 Glu 0.5 12-7.5 0.05M 100 60 3.770.75-1.8 Glu 1 12-7.5 0.05M 100 60 2.77 0.28-0.7 *% RSD varied from 5-7

Four different concentrations of NaOH (0.2, 0.1, 0.05 and 0.025M) areused for the preparation of acetol. Table 4 below shows results of thisstudy. As can be observed, when the NaOH concentration is 0.025M, theleast amount of acetol is obtained. However, as the concentrationincreases from 0.025 to 0.1M, the mass of synthesized acetol increasestoo. When the concentration of base increases to 0.2M, the amount ofacetol decreases. It is noted that the reaction solution smells of burntsugar compared to the other reactions where the base concentration islower.

TABLE 4 Effect of NaOH Concentration on synthesis of acetol % Yield wt.Wt. Temp, Time, mg base-mole Sugar gr pH NaOH C. min acetol* base Glu0.5  12-11.5 0.2M 100 60 3.35 0.67-1.6 Glu 0.5 12-8.5 0.1M 100 60 5.381.08-2.6 Glu 0.5 12-7.5 0.05M  100 60 3.77 0.75-1.8 Glu 0.5 12-6.50.025M  100 60 2.01 0.40-1.0 *% RSD varied from 5-20 (% RSD was 20 for0.2M NaOH reactions)

The effects of temperature on the synthesis of acetol using NaOH andglucose are investigated. For this purpose, different temperaturesranging from 90 to 140° C. are studied. Results for reactiontemperatures of 90, 100 and 120° C. are similar while the reaction at140° C. yields approximately 25% more acetol than the other reactionswith lower temperatures. Table 5 below shows results of this study.

TABLE 5 Effect of Reaction Temperature on synthesis of acetol % Yieldwt. Wt. Temp, Time, mg base-mole Sugar gr pH NaOH ° C. min acetol* baseGlu 0.5 12-7.0 0.05M 90 60 3.37 0.67-1.6 Glu 0.5 12-7.5 0.05M 100 603.77 0.75-1.8 Glu 0.5 12-7.5 0.05M 120 60 3.23 0.65-1.6 Glu 0.5 12-6.80.05M 140 60 4.58 0.92-2.2 *% RSD varied from 2-8

The effects of reaction time on the synthesis of acetol is also studied.Reactions are performed using identical conditions while reaction timesare varied from 60 to 720 minutes. Table 6 below shows the results ofthis study. The results show that with increasing reaction times, thereis no significant change in the yield of acetol.

TABLE 6 Effect of Reaction Time on synthesis of acetol % Yield wt. Wt.Temp, Time, mg base-mole Sugar gr pH NaOH ° C. min acetol* base Glu 0.512-7.5 0.05M 100 60 3.77 0.75-1.8 Glu 0.5 12-6.8 0.05M 100 120 3.240.65-1.6 Glu 0.5 12-6.8 0.05M 100 240 3.72 0.74-1.8 Glu 0.5 12-6.9 0.05M100 720 3.38 0.68-1.6 *% RSD varied from 2-6

In the last part of this study, based on results obtained from previousreactions, different reactions are performed using optimized conditions.In reaction A, 1 gram of glucose is reacted with 0.05M NaOH under refluxat 100° C., while the pH is kept constant around 11-12. This reactioncontinues for 120 minutes until the pH remains constant and does notchange. The yield of acetol is 6.7 mg. In reaction B, a Parr vessel isused at 100° C. to react 0.5 gram of HFTS with 25 mL of 0.05M NaOH for60 minutes. The acetol yield is 3.42 mg. When the same reaction isperformed under optimized conditions, (0.1M NaOH and 140° C. for 60 min)the acetol yield increases by 100% to 6.69 mg (Rxn D). Similar resultsare obtained when optimized conditions are applied to 0.5 grams ofglucose (Rxn C). The acetol yield increases to 8.2 mg. Using identicalreaction conditions, but using 50% less glucose (0.25 gram), the yieldof acetol decreases to 5.32 mg.

TABLE 7 Effect of Different Reaction Conditions on synthesis of acetol %Yield wt. Wt. Temp, Time, mg base-mole Sugar gr pH NaOH ° C. min acetolbase Rxn A Reflux Glu 1   12.0-11.0⁺ 0.05M  100 120 6.70 0.67-1.6 Rxn BParr HFTS* 0.5 12.0-7.5 0.05M  100 60 3.42 0.68-1.7 Rxn C Parr Glu 0.512.5-6.5 0.1M 140 60 8.20 1.64-4.0 Rxn D Parr HFTS 0.5 12.5-6.7 0.1M 14060 6.69 1.34-3.3 Rxn E Parr Glu 0.25 12.0-8.5 0.1M 140 60 5.32 2.13-5.2*“HFTS” stands for high fructose tobacco syrup ⁺pH was kept constantaround 12

In summary, different parameters such as sugar type, reactiontemperature, reaction time, pH, base and sugar concentrations areoptimized for the synthesis of acetol from reaction sugars and sodiumhydroxide. It is demonstrated that the highest yield of acetol can beobtained when the reaction is performed at 140° C. using 0.1M sodiumhydroxide and 0.5 grams of glucose for 60 minutes. It is noticed thatthe pH of the reaction can be changed rapidly (within 10 minutes) from12 to 6.5 if the base concentration is low or the sugar concentration ishigh. Also, it is noted that at high temperatures (140° C.), thereaction product contains small amounts of acetoin (along with theacetol). It is important to note that hydroxy ketones (acetoin) havebeen prepared from glucose using biotechnical approaches with yieldgreater than 90%. However, published chemical conversions of glucose tohydroxy ketones are much less at −9%.

Example 7

Pyrazines are produced using glucose as a carbon source.

As illustrated in Example 6 above, acetol can be synthesized via areaction of 0.1 N NaOH and glucose using optimum conditions (0.5 gramglucose reacted with 25 mL of 0.1 N NaOH at 140° C. for 60 min). Theyield of acetol was approximately 2% based on the glucose weight.

A publication by Nodzu (R. Nodzu, On the action of phosphate uponhexoses, The formation of acetol from glucose in acidic solution ofpotassium phosphate. Bull Chem. Soc. Japan, 10, 122-130, 1935, which isherein incorporated by reference) demonstrated that acetol with 4% yield(based on the weight of the glucose) can by synthesized from a reactionof a 40% phosphate buffer solution with a pH of 6.5-6.8 with glucose ata temperature of 100-120° C. They demonstrated that a higher yield ofacetol can be obtained at a pH of 7.0-7.1 and as pH decreased, the yieldof acetol decreased.

This Example first illustrates the preparation of acetol via a reactionof 0.1 N NaOH and glucose under optimized conditions. A method toisolate the acetol from the reaction mixture is then illustrated.Finally, it is demonstrated that the above mixture, without theisolation of acetol acetoin, can be reacted with NH₄OH to synthesizedifferent branched pyrazines.

Glucose, 1-OH-acetone (acetol), 1-OH-2-butanone (acetoin), sodiumhydroxide, di-sodium hydrogen phosphate/sodium hydroxide buffer solutionpH=12, potassium phosphate dibasic, potassium phosphate monobasic,sodium chloride, sodium sulfate anhydrous, hydrochloric acid, methanol,and dichloromethane are obtained from Sigma-Aldrich (St. Louis, Mo.).

Synthesis of acetol is performed in a 40 mL or 1.5 L Parr vessel. Tosynthesize acetol, 0.5 grams of glucose is mixed for every 25 mL of 0.1N NaOH or buffer solution. Each reaction is stirred and heated at 140°C. for a period of 60 min. The initial pH levels for all reactions areapproximately 12 and no adjustments are made during the reaction. Afterthe completion of each reaction, the mixture is cooled and the quantityof both acetol and sugar are measured using GC and HPLC. For acetolquantification, 25 mL of solution is spiked with 1 mg of acetoin as aninternal standard and extracted with 30-35 mL of DCM. Next, theextracted DCM solution is dried over sodium sulfate and analyzed viaGC/FID for quantification.

The synthesis of pyrazines is performed in the same Parr vessel.Reactions are performed by reacting the acetol synthesized from thereaction of sugar and 0.1 N NaOH. In this reaction, every 25 mL ofsolution is reacted either with 0.25, 0.5 or 1 mL of NH₄OH. Reactionsare obtained at 120 or 140° C. for a period of 17 hours. Next, eachreaction mixture is cooled and the pyrazines are extracted andquantified. In each extraction 0.25 mg of d₆-2 methyl pyrazine is addedas an internal standard and the solution is extracted with 30-35 mL ofDCM as a solvent. Next, the DCM solution is dried over sodium sulfateand analyzed via GC/MS for quantification. Extracted ions are used toquantify each pyrazine.

All GC/MS analyses are performed using the same equipment and operatingparameters as those used in Example 1 above. MS Wiley library is used toidentify each pyrazine. For quantitative analysis, the calibration curveis prepared using a concentration of acetol and a response factor ratioof acetol/acetoin. It is noted here that acetoin was not detected in anyreactions performed at 90, 100 and 120° C. However, acetoin residue isfound in the reaction where the temperature is set to 140° C. Therefore,acetoin concentrations are determined in the reaction and this value isaccounted for in all calculations.

All HPLC/RI separations are performed using Sugar-Pak (300×6.5 mm)columns from Waters (Milford, Mass.). An Agilent 1100 series HPLCequipped with a quaternary pump, refractive index (RI) detectors,auto-sampler, and oven heater set to 80° C. is employed. The isocraticmobile phase for analysis of sugar is 0.005% EDTA disodium dihydrate.The flow rate is set at 0.5 mL/min for these analyses.

First, acetol is synthesized using a buffer solution. Due to rapidchanges in the pH of a solution in a reaction of glucose with 0.1 N NaOHsolution for synthesis of acetol, a buffer solution with a pH of 12(purchased from Sigma Aldrich) is used in the synthesis of acetolinstead of a 0.1 N NaOH solution. In this study, 25 mL of di-sodiumhydrogen phosphate/sodium hydroxide buffer solution pH=12 is reactedwith 0.25 or 0.5 gram of glucose at 140° C. for 60 minutes. Results showthat the concentration of acetol in the reaction product is much higher(2×) when a buffer solution is used to perform the synthesis. Table 8below shows the results of this study.

TABLE 8 Effect of glucose concentration on preparation of acetol usingbasic buffer solution from Sigma Aldrich Buffer Rxn, Acetol, % YieldGlucose Volume Time Temp., mass based on Solution mg mL pH Min ° C. mgGlucose Buffer 0.25 25 12 60 140 6.53 5.2 Buffer 0.50 25 12 60 140 15.36.1

When a similar buffer is prepared (mixing 7.1 gram of Na₂HPO₄ and 1 gramof NaOH in 100 mL of H₂O with a pH of approximately 12 and capacity of0.05 N) and reacted with glucose at 140° C. for 60 minutes, the reactionmixture did not show a presence of acetol. This reaction is repeatedthree times and acetol is not found in any of the reaction products.However, when a similar buffer with the same pH but lower capacity(0.025 N) is used, acetol is found in the reaction product andconcentration of it is much higher than when 0.1 N NaOH was used. Assuch, it is determined that the buffer type, capacity, pH, and reactiontemperature have an effect on the synthesis of acetol using glucose.

Next, the acetol concentration in a reaction of glucose with 40%phosphate buffer with pH 6.5-7.0 is determined. 25 mL of a 40% potassiumphosphate buffer pH=6.5-6.8 is reacted with 0.5 or 1 gram of glucose at140° C. for 60 minutes using a Parr reactor. Table 9 below shows themass of acetol obtained from a reaction of a 40% phosphate buffer (25mL) at a pH of 6.5-6.8 with different concentrations of glucose. It isnoted here other types of hydroxy ketones such 3-OH-2-butanone and1-OH-2-butanone are also formed in this reaction. The quantity of thesehydroxy ketones is not measured. FIG. 3 shows the GC/MS analysis ofglucose reacted with a phosphate buffer at 140° C. for 60 min andextracted with DCM.

TABLE 9 Mass of Acetol obtained from reaction of 40% phosphate bufferwith different concentration of glucose Rxn, Acetol, % Yield GlucoseVolume Time Temp. mass based on Solution (g) (mL) (min) (° C.) (mg)Glucose Buff. Phosph. 0.5 25 60 140 23.57 4.7 Buff. Phosph. 0.5 25 60140 16.4 3.3 Buff. Phosph. 0.5 25 60 140 15.92 3.2 Buff. Phosph. 1 25 60140 44.3 4.4 Buff. Phosph. 1 25 60 140 44.4 4.4

Next, the glucose and acetol concentrations in 300 mL and 1 L reactionsare determined. 6 grams of glucose is reacted with 300 mL of 0.1 N NaOHfor 60 min at 140° C. After the reaction is cooled, both acetol andglucose concentrations in the solution are obtained. A small amount(0.964 mg/mL) of glucose is detected in the reactant. This correspondsto about less than 5% of the glucose that remained unreacted. Table 10below shows the concentration of acetol in this reaction. It can bedetermined that the concentration of acetol is about 12 mg for every 25mL. Two additional reactions are performed using 1 liter of 0.1 N NaOHand 20 grams of glucose using a high pressure reaction vessel. Bothreactions are performed at 130-140° C. for 60 minutes. Both glucose andacetol concentrations are determined. In both 1 liter reactions,concentrations of glucose are less than 1 mg/mL (0.945 and 0.958 mg/mL).Table 10 below also shows the concentration of acetol in each 1 literreaction. Again, the acetol concentration is about 12 mg for every 25mL.

TABLE 10 Calculated mass of Acetol from reaction of 0.1N NaOH withdifferent mass of glucose Rxn, Acetol, % Yield Glucose Volume Time Temp.mass based on Solution (g) (mL) (min) (° C.) (mg) Glucose 0.1N 6 300 60140 140.26 2.3 NaOH 0.1N 6 300 60 140 140.68 2.3 NaOH 0.1N 20 1000 60140 490.12 2.5 NaOH 0.1N 20 1000 60 140 470.08 2.4 NaOH

Next, acetol is isolated from the reaction of glucose with 0.1N NaOH.Different methods are used to isolate acetol from the reaction of 0.1 NNaOH and glucose. Previous results for the isolation of pyrazines viadistillation demonstrated that pyrazines with a boiling point of 140° C.were isolated from the reaction mixture. Therefore, a distillationapparatus set at 120-140° C. is used to isolate acetol from the reactionmixture. For this purpose, 100 mL of a reaction mixture of 0.1 N NaOHand glucose is distilled at 140° C. After the collection of 40 mL ofdistillation solution, both the distilled and the remaining materialsare extracted with DCM. GC/FID analysis shows that only 15-20% of acetolis distilled while more than 80-85% of acetol remains in the reactionmixture.

Similar results are obtained when a reaction mixture of glucose with a40% phosphate buffer at 140° C. for 60 minutes is distilled. For thisdistillation, water is continuously added to the flask during thedistillation to keep the distilled solution volume constant. After thecollection of about 10 mL solution via distillation, approximately 10 mLH₂O is added to the distillation flask to compensate for the volumelost. A total of 4×10 mL fractions are collected. Acetol is observed ineach fraction. Analysis of the remaining reaction mixture with DCM showsa presence of more acetol in the solution. The isolation of acetol canbe obtained if this distillation continues for several hours.

In a second method, column chromatography is used to isolate the acetolfrom the reaction mixture. For this purpose a 30×2.0 cm glass (or metal)column (packed up to only 12-15 cm) with C₁₈ (particulate size of 40-60μm and 90 Å pore size) is used to isolate the acetol. After washing thecolumn with methanol followed by a 0.1% FA solution, 25 mL of thereaction mixture containing acetol is passed through the column. All ofthe solution is collected during this isolation. After pushing thesolution through the column (fraction 1, 25 mL), 25 mL of deionized H₂Ois used to wash the material from the column (Fraction 2). Next, thecolumn is washed with an additional 25 mL H₂O until clean H₂O is elutedfrom the column (Fraction 3). Then the column is dried with N₂, and theremaining analytes trapped in the column are eluted with 100% MeOH(Fraction 4). All fractions are extracted with DCM and analyzed viaGC/FID. Results showed that most of the acetol is eluted with fractions1 and 2 while fractions 3 and 4 did not contain any acetol. It is notedhere that after passing the reaction mixture through the C₁₈ packing, itis very difficult to use the packing for another reaction mixturecleanup. It is very difficult to clean up the packing. In summary,isolating the hydroxy ketones from the reaction product can be timelyand expensive.

Next, pyrazines are synthesized from the reaction product of glucose andsodium hydroxide with NH₄OH, without first isolating the hydroxyketones. After the preparation of acetol via reacting 0.5 grams glucosewith 25 mL of 0.1 N NaOH at 140° C. for 60 minutes, it is determinedthat the reaction mixture contained about 10-12 mg of acetol/25 mL ofsolution. For this purpose, 25 mL of the solution is reacted with 0.5 or1 mL of NH₄OH for a period of 17-18 hours at 120-140° C. to determinethe type and the percent yield of the pyrazines. Each reaction isrepeated twice. Table 11 below shows list of pyrazines that are detectedincluding their elution time from the GC column. Results are similarwhen the volume of NH₄OH is changed from 0.5 to 1 mL.

TABLE 11 List of synthesized pyrazines Retention time Peak # Analyte5.82 1 Internal Standard 5.08 2 Pyrazine 5.82 3 2-Methylpyrazine 6.48 42,6-Dimethylpyrazine 6.56 5 2,5-Dimethylpyrazine 6.76 62,3-Dimethylpyrazine 7.178 7 2-Ethyl-5-Methylpyrazine 7.24 82-Ethyl-6-Methylpyrazine 7.4 9 2,3,5-Trimethylpyrazine 7.95 102,6-Dimethyl-3-Ethylpyrazine 8 11 2,Ethyl-2,5-Dimethylpyrazine 8.1 122,3,5,6-Tetramethylpyrazine 8.44 13 3,5-Diethyl-2-Methylpyrazine 8.63 142,5-Dimethyl-3Propylpyrazine 8.81 15 2,5-Diethyl-3,6- Dimethylpyrazine9.4 16 6,7-DiHydrocyclo-5- Methylcyclopentapyrazine 9.669 172-Isoprophenylpyrazine 9.69 18 2,3-Dimethyl-5- Isopentylpyrazine 9.89 192,5-Dimethyl-3-(3- methylbutyl)pyrazine 9.96 206,7-DiHydrocyclo-2,5-Dimethyl- 5H-cyclopentapyrazine 10.238 212-Methyl-5H-6,7- diHydrocyclopentapyrazine

Similar reactions are performed in the 1 liter scale. Two batches of 1liter solution are prepared. In each batch, 20 grams of glucose isreacted with 1000 mL of 0.1 N NaOH solution at 140° C. for 60 min. Eachsolution is cooled and the acetol concentration is measured. Next, inone reaction mixture, 20 mL of NH₄OH is added, and in a second reactionmixture, 40 mL of NH₄OH is added. Each reaction is heated at 120-130° C.for 17 hours with continuous mixing. After the reactions are cooled, 25mL of each reaction is spiked with an internal standard(d₆-2-methylpyrazine) and extracted with DCM to determine theconcentration and distribution of each pyrazine. Each remaining reactionsolution is distilled and collected separately. Approximately 125 mL ofdistilled solution is collected for every 500 mL of reaction mixture.Next, the distilled solution (10 mL) is extracted with DCM andquantified via GC/MS. The total mass of pyrazines is higher than if thesolution had not been extracted with DCM, which is due to a higherconcentration of pyrazines in the distilled solution. The distributionof pyrazines is almost identical in both extracts. It is noted that both2-methylpyrazine and pyrazine are detected in large quantities in allreactions when NH₄OH is reacted with the reaction mixture of glucosewith 0.1 N NaOH. No pyrazines are detected in the extract of theremaining solution after distillation.

Next, pyrazines are synthesized from the reaction product of glucose andphosphate buffer with NH₄OH, without first isolating the hydroxyketones. 25 mL of the reaction mixture of a phosphate buffer withpH=6.5-6.8 and glucose (0.5 or 1 gram, solutions A and B, respectively)is reacted with 1 mL of NH₄OH at 140° C. for 17 hours. After eachreaction is cooled down, 0.25 mg of deuterated 2-methylpyrazine(internal standard) is added to each reaction mixture and the solutionsare extracted with DCM. Each extract is analyzed using GC/MS and themass of pyrazines and their percent distribution is calculated. It isnoted that both pyrazine and 2-methylpyrazine are not synthesized inthese reactions. Also, the total mass of pyrazines is higher by 20% insolution B when 1 gram of glucose is reacted with a phosphate buffer.Without being limited by theory, it is believed that this is due to ahigher concentration of acetol in solution B compared to solution A.FIG. 4 shows the GC/MS analysis of pyrazines extracted using DCM from areaction mixture of solution B (1 gram of glucose reacted with 25 mL of40% phosphate buffer at 140° C. for 60 min) with 1 mL of NH₄OH at 140°C. for 17 hours. For comparison, FIG. 5 shows the GC/MS analysis ofpyrazines extracted using DCM from 25 mL of a reaction mixture of 0.1 NNaOH reacted with 0.5 gram of glucose at 140° C. for 60 min and thenreacted with 1 mL of NH4OH at 140° C. In this reaction, both pyrazineand 2-methylpyrazine are detected.

In summary, acetol (and other hydroxy ketones) can be isolated from areaction mixture of glucose and 0.1 N NaOH using both distillation andcolumn chromatography. Distillation is very time and energy consumingwhile chromatography can become very expensive. However, it is possibleto synthesize pyrazines without the isolation of acetol. When the acetolprepared from the reaction of 0.1 N NaOH and glucose reacted with NH₄OHat 140° C. for 17 hours, an array of pyrazines were produced. Thepyrazines were isolated via distillation. It is noted that pyrazine and2-methylpyrazine were synthesized during this process. However, when theacetol prepared from the mixture of glucose and phosphate buffer reactedwith NH₄OH, similar pyrazines were produced with a similar distribution,but no pyrazine or 2-methyl pyrazine was detected in the reactionmixture.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

What is claimed is:
 1. A method of forming pyrazines comprising:receiving a reaction solution comprising at least one alpha-hydroxyketone and at least one nitrogen source; adding at least one free aminoacid to the reaction solution; heating the reaction solution to areaction temperature and holding the reaction solution at the reactiontemperature for a time sufficient to produce a reaction productcomprising at least one substituted pyrazine; and isolating the at leastone substituted pyrazine from the reaction product, wherein the step ofisolating the at least one substituted pyrazine from the reactionproduct comprises at least one of liquid-liquid extraction of thereaction product, liquid-solid extraction of the reaction product, andsimple distillation of the reaction product.
 2. The method of claim 1,wherein the at least one hydroxy ketone comprises acetol.
 3. The methodof claim 2, wherein the at least one substituted pyrazine is selectedfrom the group consisting of: 2,3-dimethylpyrazine;2,6-dimethylpyrazine; 2-ethyl-6-methylpyrazine;2-ethyl-5-methylpyrazine; trimethylpyrazine;2,5-dimethyl-3-ethylpyrazine; 2-ethyl-3,5-dimethylypyrazine;tetramethylpyrazine; 2,5-dimethyl-3-propenylpyrazine;2,3,5-trimethyl-6-isopropylpyrazine; 2-acetyl-4,5-dimethylpyrazine;3,5-dimethyl-2-methylpropylpyrazine, and combinations thereof.
 4. Themethod of claim 1, wherein the at least one hydroxy ketone comprisesacetoin.
 5. The method of claim 4, wherein the at least one substitutedpyrazine is tetramethylpyrazine.
 6. The method of claim 1, wherein theat least one hydroxy ketone comprises 1-hydroxy-2-butanone.
 7. Themethod of claim 6, wherein the at least one substituted pyrazine isselected from the group consisting of: 2,6-diethylpyrazine;2,5-diethylpyrazine; 2- ethyl-3,5,6-trimethylpyrazine;3,5-dimethyl-2-(n-propyl)pyrazine; 3,6-dimethyl-2-(n-propyl)pyrazin;2,5-3-methylpyrazine; 2,3-diethyl-5-methylpyrazine; 2,3-diethyl-5,6-dimethylpryazine; trans-3-methyl-2-(n-propyl)-6-(butenyl)pyrazine;2,5-dimethyl-3-ethylpyrazine; and combinations thereof.
 8. The method ofclaim 1, wherein the nitrogen source is selected from the groupconsisting of amino acids, ammonium ions, and combinations thereof. 9.The method of claim 1, further comprising adding at least one aldehydeto the reaction solution.
 10. The method of claim 1, wherein thereaction temperature is about 90° C. to about 150° C.
 11. A method offorming pyrazines comprising: receiving a reaction solution comprisingat least one alpha-hydroxy ketone and at least one nitrogen source;adding at least one free amino acid to the reaction solution; andheating the reaction solution to a reaction temperature and holding thereaction solution at the reaction temperature for a time sufficient toproduce a reaction product comprising at least one substituted pyrazine;wherein the at least one substituted pyrazine is disubstituted,trisubstituted, or tetrasubstituted.
 12. The method of claim 1, whereinthe at least one substituted pyrazine is disubstituted.
 13. The methodof claim 1, wherein the at least one substituted pyrazine is trisubstituted.
 14. The method of claim 1, wherein at least one substitutedpyrazine is tetrasubstituted.
 15. The method of claim 1, wherein the atleast one substituted pyrazine comprises at least one substituent grouphaving 2 or more carbon atoms.
 16. The method of claim 1, wherein the atleast one substituted pyrazine comprises at least one substituent grouphaving 3 or more carbon atoms.
 17. The method of claim 1, wherein thereaction product is substantially free of the molecule pyrazine and themolecule methylpyrazine.
 18. The method of claim 1, further comprisingincorporating the at least one substituted pyrazine into a tobaccoproduct.
 19. The method of claim 18, wherein the tobacco product is asmoking article or a smokeless tobacco product.
 20. A method of formingpyrazines comprising: receiving a reaction solution comprising at leastone alpha-hydroxy ketone and at least one nitrogen source; and heatingthe reaction solution to a reaction temperature and holding the reactionsolution at the reaction temperature for a time sufficient to produce areaction product comprising at least one substituted pyrazine; whereinthe at least one substituted pyrazine is disubstituted.
 21. The methodof claim 1, wherein the at least one free amino acid is selected fromthe group consisting of serine, alanine, leucine, isoleucine, valine,threonine, phenylalanine, and combinations thereof.
 22. A method offorming pyrazines comprising: receiving a reaction solution comprisingat least one alpha-hydroxy ketone and at least one nitrogen source;adding at least one free amino acid to the reaction solution; andheating the reaction solution to a reaction temperature and holding thereaction solution at the reaction temperature for a time sufficient toproduce a reaction product comprising at least one substituted pyrazine;wherein the at least one hydroxy ketone comprises acetol; and whereinthe at least one substituted pyrazine is selected from the groupconsisting of: 2,3-dimethylpyrazine; 2,6-dimethylpyrazine;2-ethyl-6-methylpyrazine; 2-ethyl-5-methylpyrazine; trimethylpyrazine;2,5dimethyl-3-ethylpyrazine; 2-ethyl-3,5-dimethylypyrazine;tetramethylpyrazine; 2,5-dimethyl-3-propenylpyrazine;2,3,5-trimethyl-6-isopropylpyrazine; 2-acetyl-4,5-dimethylpyrazine;3,5-dimethyl-2-methylpropylpyrazine, and combinations thereof.
 23. Amethod of forming pyrazines comprising: receiving a reaction solutioncomprising at least one alpha-hydroxy ketone and at least one nitrogensource; adding at least one free amino acid to the reaction solution;and heating the reaction solution to a reaction temperature and holdingthe reaction solution at the reaction temperature for a time sufficientto produce a reaction product comprising at least one substitutedpyrazine; wherein the at least one hydroxy ketone comprises acetoin. 24.A method of forming pyrazines comprising: receiving a reaction solutioncomprising at least one alpha-hydroxy ketone and at least one nitrogensource; adding at least one free amino acid to the reaction solution;and heating the reaction solution to a reaction temperature and holdingthe reaction solution at the reaction temperature for a time sufficientto produce a reaction product comprising at least one substitutedpyrazine; wherein the at least one hydroxy ketone comprises1-hydroxy-2-butanone.
 25. The method of claim 11, wherein the at leastone substituted pyrazine is selected from the group consisting of:2,6-dimethylpyrazine; 2,5-dimethylpyrazine; 2-ethyl-5-methylpyrazine;2-ethyl-6-methylpyrazine; 2,3,5-trimethylpyrazine;2-ethyl-3,5-dimethylpyrazine; 2-ethyl-2,5-dimethylpyrazine;2,3,5,6-tetramethylpyrazine; 2,3,5-trimethyl-6-ethylpyrazine;2,6-dimethyl-3-propylpyrazine; 2,5-diethyl-3,6-dimethylpyrazine;2,6-dimethyl-3-(2-methylbutyl)pyrazine;2,5-dimethyl-3-(2-methylbutyl)pyrazine;2,5-dimethyl-3-(3-methylbutyl)pyrazine; 2,5-dimethyl-3-propylpyrazine;2,5-dimethyl-3-cis-propenylpyrazine; 2-isopropenyl-3,6-dimethylpyrazine;2-(2-methylpropyl)-3,5-dimethylpyrazine;2,6-dimethyl-3-isobutylpyrazine;2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine;trimethylpyrazine; 2,5-dimethyl-3-ethylpyrazine; tetramethylpyrazine;2,3-diethyl-5-methylpyrazine; 2,5-dimethyl-3-propenylpyrazine;2,3,5-trimethyl-6-isopropylpyrazine; 2-acetyl-4,5-dimethylpyrazine;3,5-dimethyl-2-methylpropylpyrazine; 2,6-diethylpyrazine;2,5-diethylpyrazine; 2-ethyl-3,5,6-trimethylpyrazine;3,5-dimethyl-2-(n-propyl)pyrazine; 3,6-dimethyl-2-(n-propyl)pyrazine;2,5-diethyl-3-methylpyrazine; 2,3-diethyl-5,6-dimethylpryazine;trans-3-methyl-2-(n-propyl)-6-(butenyl)pyrazine;2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrazine; and2,5-dimethyl-3-ethylpyrazine; and combinations thereof.